Dual-acting pyrazole antihypertensive agents

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
     
       
         
         
             
             
         
       
     
     wherein: Ar, Z, R 3 , R 4  and R 5  are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have AT 1  receptor antagonist activity and neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and process and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/223,472, filed on Jul. 7, 2009; the entire disclosure of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds having angiotensin IItype 1 (AT₁) receptor antagonist activity and neprilysin-inhibitionactivity. The invention also relates to pharmaceutical compositionscomprising such compounds, processes and intermediates for preparingsuch compounds and methods of using such compounds to treat diseasessuch as hypertension.

2. State of the Art

The aim of antihypertensive therapy is to lower blood pressure andprevent hypertension-related complications such as myocardialinfarction, stroke, and renal disease. For patients with uncomplicatedhypertension (that is, no risk factors, target organ damage, orcardiovascular disease), it is hoped that reducing blood pressure willprevent development of cardiovascular and renal comorbidities,conditions that exist at the same time as the primary condition in thesame patient. For those patients with existing risk factors orcomorbidities, the therapeutic target is the slowing of comorbid diseaseprogression and reduced mortality.

Physicians generally prescribe pharmacological therapies for patientswhose blood pressure cannot be adequately controlled by dietary and/orlifestyle modifications. Commonly used therapeutic classes act topromote diuresis, adrenergic inhibition, or vasodilation. A combinationof drugs is often prescribed, depending upon what comorbidities arepresent.

There are five common drug classes used to treat hypertension:diuretics, which include thiazide and thiazide-like diuretics such ashydrochlorothiazide, loop diuretics such as furosemide, andpotassium-sparing diuretics such as triamterene; β₁ adrenergic receptorblockers such as metoprolol succinate and carvedilol; calcium channelblockers such as amlodipine; angiotensin-converting enzyme (ACE)inhibitors such as captopril, benazepril, enalapril, enalaprilat,lisinopril, quinapril, and ramipril; and AT₁ receptor antagonists, alsoknown as angiotensin II type 1 receptor blockers (ARBs), such ascandesartan cilexetil, eprosartan, irbesartan, losartan, olmesartanmedoxomil, telmisartan, and valsartan. Combinations of these drugs arealso administered, for example, a calcium channel blocker (amlodipine)and an ACE inhibitor (benazepril), or a diuretic (hydrochlorothiazide)and an ACE inhibitor (enalapril). All of these drugs, when usedappropriately, are effective in the treatment of hypertension.Nevertheless, both efficacy and tolerability should be further improvedin new drugs targeting hypertension. Despite the availability of manytreatment options, the recent National Health And Nutrition ExaminationSurvey (NHANES) demonstrated that only about 50% of all treated patientswith hypertension achieve adequate blood pressure control. Furthermore,poor patient compliance due to tolerability issues with availabletreatments further reduces treatment success.

In addition, each of the major classes of antihypertensive agents havesome drawbacks. Diuretics can adversely affect lipid and glucosemetabolism, and are associated with other side effects, includingorthostatic hypotension, hypokalemia, and hyperuricemia. Beta blockerscan cause fatigue, insomnia, and impotence; and some beta blockers canalso cause reduced cardiac output and bradycardia, which may beundesirable in some patient groups. Calcium channel blockers are widelyused but it is debatable as to how effectively these drugs reduce fataland nonfatal cardiac events relative to other drug classes. ACEinhibitors can cause coughing, and rarer side effects include rash,angioedema, hyperkalemia, and functional renal failure. AT₁ receptorantagonists are equally effective as ACE inhibitors but without the highprevalence of cough.

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is anendothelial membrane bound Zn²⁺ metallopeptidase found in many tissues,including the brain, kidney, lungs, gastrointestinal tract, heart, andperipheral vasculature. NEP is responsible for the degradation andinactivation of a number of vasoactive peptides, such as circulatingbradykinin and angiotensin peptides, as well as the natriureticpeptides, the latter of which have several effects includingvasodilation and diuresis. Thus, NEP plays an important role in bloodpressure homeostasis. NEP inhibitors have been studied as potentialtherapeutics, and include thiorphan, candoxatril, and candoxatrilat. Inaddition, compounds have also been designed that inhibit both NEP andACE, and include omapatrilat, gempatrilat, and sampatrilat. Referred toas vasopeptidase inhibitors, this class of compounds are described inRobl et al. (1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

There may be an opportunity to increase anti-hypertensive efficacy whencombining AT₁ receptor antagonism and NEP inhibition, as evidenced byAT₁ receptor antagonist/NEP inhibitor combinations described in WO9213564 to Darrow et al (Schering Corporation); US20030144215 to Ksanderet al.; Pu et al., Abstract presented at the Canadian CardiovascularCongress (October 2004); and Gardiner et al. (2006) JPET 319:340-348;and WO 2007/045663 (Novartis AG) to Glasspool et al.

Recently, WO 2007/056546 (Novartis AG) to Feng et al. has describedcomplexes of an AT₁ receptor antagonist and a NEP inhibitor, where anAT₁ receptor antagonist compound is non-covalently bound to a NEPinhibitor compound, or where the antagonist compound is linked vianon-covalent bonding to the inhibitor compound.

In spite of the advances in the art, there remains a need for a highlyefficacious monotherapy with multiple mechanisms of action leading tolevels of blood pressure control that can currently only be achievedwith combination therapy. Thus, although various hypertensive agents areknown, and administered in various combinations, it would be highlydesirable to provide compounds having both AT₁ receptor antagonistactivity and NEP inhibition activity in the same molecule. Compoundspossessing both of these activities are expected to be particularlyuseful as therapeutic agents since they would exhibit antihypertensiveactivity through two independent modes of action while having singlemolecule pharmacokinetics.

In addition, such dual-acting compounds are also expected to haveutility to treat a variety of other diseases that can be treated byantagonizing the AT₁ receptor and/or inhibiting the NEP enzyme.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess AT₁ receptor antagonist activity and neprilysin (NEP) enzymeinhibition activity. Accordingly, compounds of the invention areexpected to be useful and advantageous as therapeutic agents fortreating conditions such as hypertension and heart failure.

One aspect of the invention relates to a compound of formula I:

where: Z is a pyrazole selected from:

Ar is selected from:

R¹ is selected from —SO₂NHC(O)R^(1a), tetrazolyl, —C(O)OR^(1b),

where R^(1a) is —C₁₋₆alkyl, —C₀₋₆alkylene-OR, —C₃₋₇cycloalkyl,—C₀₋₅alkyleneNR^(1b)R^(1b), pyridyl, isoxazolyl, methylisoxazolyl,pyrrolidinyl, morpholinyl, and phenyl optionally substituted with halo;where each R^(1b) is independently selected from H and —C₁₋₆alkyl;

a is 0, 1, or 2; R² is F;

R³ is selected from —C₂₋₅alkyl and —O—C₁₋₅alkyl;

R⁴ is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OR^(4d)), and —CH(R^(4b))C(O)OR^(4c); where R^(4a) isH or —C(O)—C₁₋₆alkyl; R^(4b) is H or —OH; R^(4c) is H, —C₁₋₆alkyl,—C₀₋₆alkylenemorpholine, —CH₂OC(O)O—C₁₋₆alkyl, —CH(CH₃)OC(O)O—C₁₋₆alkyl,—CH(CH₃)OC(O)O—C₃₋₇cycloalkyl, or:

R^(4d) is H or —C(O)—R^(4e); and R^(4e) is —C₁₋₆alkyl, —C₁₋₆alkyl-NH₂ oraryl; and

R⁵ is selected from —C₁₋₆alkyl, —CH₂-furanyl, —CH₂-thiophenyl, benzyl,and benzyl substituted with one or more halo, —CH₃, or —CF₃ groups;

where each ring in Ar is optionally substituted with 1 to 3 substituentsindependently selected from —OH, —C₁₋₆alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,—CN, halo, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, —S(O)—C₁₋₆alkyl,—S(O)₂—C₁₋₄alkyl, -phenyl, —NO₂, —NH₂, —NH—C₁₋₆alkyl and —N(C₁₋₆alkyl)₂,wherein each alkyl, alkenyl and alkynyl is optionally substituted with 1to 5 fluoro atoms;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents such as diuretics, β₁ adrenergic receptor blockers, calciumchannel blockers, angiotensin-converting enzyme inhibitors, AT₁ receptorantagonists, neprilysin inhibitors, non-steroidal anti-inflammatoryagents, prostaglandins, anti-lipid agents, anti-diabetic agents,anti-thrombotic agents, renin inhibitors, endothelin receptorantagonists, endothelin converting enzyme inhibitors, aldosteroneantagonists, angiotensin-converting enzyme/neprilysin inhibitors,vasopressin receptor antagonists, and combinations thereof. Accordingly,in yet another aspect of the invention, a pharmaceutical compositioncomprises a compound of the invention, a second therapeutic agent, and apharmaceutically acceptable carrier. Another aspect of the inventionrelates to a combination of active agents, comprising a compound of theinvention and a second therapeutic agent. The compound of the inventioncan be formulated together or separately from the additional agent(s).When formulated separately, a pharmaceutically acceptable carrier may beincluded with the additional agent(s). Thus, yet another aspect of theinvention relates to a combination of pharmaceutical compositions, thecombination comprising: a first pharmaceutical composition comprising acompound of the invention and a first pharmaceutically acceptablecarrier; and a second pharmaceutical composition comprising a secondtherapeutic agent and a second pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a kit containing suchpharmaceutical compositions, for example where the first and secondpharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess both AT₁ receptor antagonist activityand NEP enzyme inhibition activity, and are therefore expected to beuseful as therapeutic agents for treating patients suffering from adisease or disorder that is treated by antagonizing the AT₁ receptorand/or inhibiting the NEP enzyme. Thus, one aspect of the inventionrelates to a method of treating patients suffering from a disease ordisorder that is treated by antagonizing the AT₁ receptor and/orinhibiting the NEP enzyme, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method of treating hypertension orheart failure, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention. Still another aspect ofthe invention relates to a method for antagonizing an AT₁ receptor in amammal comprising administering to the mammal, an AT₁receptor-antagonizing amount of a compound of the invention. Yet anotheraspect of the invention relates to a method for inhibiting a NEP enzymein a mammal comprising administering to the mammal, a NEPenzyme-inhibiting amount of a compound of the invention.

Compounds of the invention that arc of particular interest include thosethat exhibit an inhibitory constant (pK_(i)) for binding to an AT₁receptor greater than or equal to about 5.0; in particular those havinga pK_(i) greater than or equal to about 6.0; in one embodiment thosehaving a pK_(i) greater than or equal to about 7.0; more particularlythose having a pK_(i) greater than or equal to about 8.0; and in yetanother embodiment, those having a pK_(i) within the range of about8.0-10.0. Compounds of particular interest also include those having aNEP enzyme inhibitory concentration (pIC₅₀) greater than or equal toabout 5.0; in one embodiment those having a pIC₅₀ greater than or equalto about 6.0; in particular those having a pIC₅₀ greater than or equalto about 7.0; and most particularly those having a pIC₅₀ within therange of about 7.0-10.0. Compounds of further interest include thosehaving a pK_(i) for binding to an AT₁ receptor greater than or equal toabout 7.5 and having a NEP enzyme pIC₅₀ greater than or equal to about7.0.

Since compounds of the invention possess AT₁ receptor antagonistactivity and NEP inhibition activity, such compounds are also useful asresearch tools. Accordingly, one aspect of the invention relates to amethod of using a compound of the invention as a research tool, themethod comprising conducting a biological assay using a compound of theinvention. Compounds of the invention can also be used to evaluate newchemical compounds. Thus another aspect of the invention relates to amethod of evaluating a test compound in a biological assay, comprising:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include an AT₁ receptorbinding assay and a NEP enzyme inhibition assay. Still another aspect ofthe invention relates to a method of studying a biological system orsample comprising an AT₁ receptor, a NEP enzyme, or both, the methodcomprising: (a) contacting the biological system or sample with acompound of the invention; and (b) determining the effects caused by thecompound on the biological system or sample.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention.Accordingly, another aspect of the invention relates to a process ofpreparing compounds of the invention comprising the step of coupling acompound of formula 1 with a compound of formula 2:

where: Ar* represents Ar—R¹*, where R¹* is R¹ or a protected form of R¹;and R⁴* represents R⁴ or a protected form of R⁴; and optionallydeprotecting the product when R¹* is a protected form of R¹ and/or R⁴*is a protected form of R⁴. Another aspect of the invention relates to aprocess of preparing a pharmaceutically acceptable salt of a compound offormula I, comprising contacting a compound of formula I in free acid orbase form with a pharmaceutically acceptable base or acid. In otheraspects, the invention relates to products prepared by any of theprocesses described herein, as well as novel intermediates used in suchprocess. In one aspect of the invention novel intermediates have formulaV, VI or VII, as defined herein.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension or heart failure. Anotheraspect of the invention relates to use of a compound of the inventionfor antagonizing an AT₁ receptor or for inhibiting a NEP enzyme in amammal. Still another aspect of the invention relates to the use of acompound of the invention as a research tool. Other aspects andembodiments of the invention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas II, III, IV and V. In addition, the compounds of the inventionmay also contain several basic or acidic groups (for example, amino orcarboxyl groups) and therefore, such compounds can exist as a free base,free acid, or in various salt forms. All such salt forms are includedwithin the scope of the invention. Furthermore, the compounds of theinvention may also exist as prodrugs. Accordingly, those skilled in theart will recognize that reference to a compound herein, for example,reference to a “compound of the invention” or a “compound of formula I”includes a compound of formula I as well as pharmaceutically acceptablesalts and prodrugs of that compound unless otherwise indicated. Further,the term “or a pharmaceutically acceptable salt and/or prodrug thereof”is intended to include all permutations of salts and prodrugs, such as apharmaceutically acceptable salt of a prodrug. Furthermore, solvates ofcompounds of formula I are included within the scope of this invention.

The compounds of formula I may contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention relates to raccmicmixtures, pure stereoisomers (enantiomers or diastereomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the term “compound offormula I” is intended to include all possible stereoisomers of thecompound. Similarly, when a particular stereoisomer is shown or namedherein, it will be understood by those skilled in the art that minoramounts of other stereoisomers may be present in the compositions of theinvention unless otherwise indicated, provided that the utility of thecomposition as a whole is not eliminated by the presence of such otherisomers. Individual enantiomers may be obtained by numerous methods thatare well known in the art, including chiral chromatography using asuitable chiral stationary phase or support, or by chemically convertingthem into diastereomers, separating the diastereomers by conventionalmeans such as chromatography or recrystallization, then regenerating theoriginal enantiomers. Additionally, where applicable, all cis-trans orE/Z isomers (geometric isomers), tautomeric forms and topoisomeric formsof the compounds of the invention are included within the scope of theinvention unless otherwise specified.

One possible chiral center could be present at the carbon on the —CHR⁴R⁵group, when R⁵ is a group such as —C₁₋₆alkyl, for example —CH₂CH(CH₃)₂.This chiral center is present at the carbon atom indicated by the symbol*:

In one embodiment of the invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, compounds offormula I have the (R) configuration at the carbon atom identified bythe symbol * or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another embodiment, the carbonatom identified by the symbol * has the (S) configuration. In thisembodiment, compounds of formula I have the (S) configuration at thecarbon atom identified by the symbol * or are enriched in astereoisomeric form having the (S) configuration at this carbon atom.

The compounds of the invention can also have two chiral centers on the—CHR⁴R⁵ group, for example when R⁴ is —CH(OH)C(O)OH and R⁵ is benzyl.These chiral centers are present at the carbons atom indicated by thesymbols * and **:

In such cases, four possible diastereomers can exist. For example, bothcarbon atoms can have the (R) configuration, and in such an embodiment,compounds of formula I have the (R) configuration at the carbon atomsidentified by the symbols * and ** or are enriched in a stereoisomericform having the (R,R) configuration at these atoms. In anotherembodiment, both carbon atoms can have the (S) configuration, and insuch an embodiment, compounds of formula I have the (S,S) configurationat the carbon atoms identified by the symbols * and ** or are enrichedin a stereoisomeric form having the (S) configuration at these atoms. Inyet another embodiment, the carbon atom identified by the symbol * canhave the (S) configuration and the carbon atom identified by the symbol** can have the (R) configuration, and in such an embodiment, compoundsof formula I have the (S,R) configuration at the carbon atoms identifiedby the symbols * and ** or are enriched in a stereoisomeric form havingthe (S,R) configuration at these atoms. In still another embodiment, thecarbon atom identified by the symbol * can have the (R) configurationand the carbon atom identified by the symbol ** can have the (S)configuration, and in such an embodiment, compounds of formula I havethe (R,S) configuration at the carbon atoms identified by the symbols *and ** or are enriched in a stereoisomeric form having the (R,S)configuration at these atoms.

In some cases, in order to optimize the therapeutic activity of thecompounds of the invention, for example, as hypertensive agents, it maybe desirable that the carbon atom identified by the symbols * and/or **have a particular (R), (S), (R,R), (S,S), (S,R), or (R,S) configuration.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F.

The compounds of formula I have been found to possess AT₁ receptorantagonizing activity and NEP enzyme inhibition activity. Among otherproperties, such compounds are expected to be useful as therapeuticagents for treating diseases such as hypertension. By combining dualactivity into a single compound, double therapy can be achieved, thatis, AT₁ receptor antagonist activity and NEP enzyme inhibition activitycan be obtained using a single active component. Since pharmaceuticalcompositions containing one active component are typically easier toformulate than compositions containing two active components, suchsingle-component compositions provide a significant advantage overcompositions containing two active components. In addition, certaincompounds of the invention have also been found to be selective forinhibition of the AT₁ receptor over the angiotensin II type 2 (AT₂)receptor, a property that may have therapeutic advantages.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one aspect, the invention relates to compounds of formula I:

Z represents a pyrazole selected from:

Thus, the compounds of the invention can also be depicted as formulas IIand III:

Ar represents an aryl group selected from:

The integer “a” is 0, 1, or 2, and the R² group is fluoro. Exemplaryfluoro-substituted Ar moieties include:

Each ring in Ar may also be substituted with 1 to 3 substituentsindependently selected from —OH, —C₁₋₆alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,—CN, halo, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, —S(O)—C₁₋₆alkyl,—S(O)₂—C₁₋₄alkyl, -phenyl, —NO₂, —NH₂, —NH—C₁₋₆alkyl and —N(C₁₋₆alkyl)₂,wherein each alkyl, alkenyl and alkynyl is optionally substituted with 1to 5 fluoro atoms.

R¹ is selected from —SO₂NHC(O)R^(1a), tetrazolyl, —C(O)OR^(1b),

The R^(1a) moiety is —C₁₋₆alkyl, —C₀₋₆alkylene-OR, —C₃₋₇cycloalkyl,—C₀₋₅alkylene-NR^(1b)R^(1b), pyridyl, isoxazolyl, methylisoxazolyl,pyrrolidinyl, morpholinyl, and phenyl optionally substituted with halo.Each R^(1b) is independently selected from H and —C₁₋₆alkyl.

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) is—C₁₋₆alkyl. Examples of this embodiment include —SO₂NHC(O)CH₃ andSO₂NHC(O)CH₂CH₃. In one particular embodiment, R¹ is tetrazolyl such as1H-tetrazol-5-yl or 5H-tetrazol-5-yl.

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) is—C₀₋₆alkylene-OR. Examples of this embodiment include —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃, and—SO₂NHC(O)(CH₂)₂OCH₃.

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is —C₃₋₇cycloalkyl. Examples of this embodiment include—SO₂NHC(O)-cyclopropyl. In another particular embodiment, R¹ is—SO₂NHC(O)R^(1a), where R^(1a) is —C₀₋₅alkylene-NR^(1b)R^(1b). Examplesof this embodiment include SO₂NHC(O)NH(CH₃), —SO₂NHC(O)N(CH₃)₂,—SO₂NHC(O)NH(CH₂CH₃), and —SO₂NHC(O)C(CH₃)₂NH₂.

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is pyridyl, for example, —SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-3-pyridyl, or—SO₂NHC(O)-4-pyridyl. The term “pyridyl” means a heterocyclic compoundof the formula:

which is bonded to any available point of attachment and includes:

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is isoxazolyl, for example, —SO₂NHC(O)-3-isoxazolyl,—SO₂NHC(O)-4-isoxazolyl, and —SO₂NHC(O)-5-isoxazolyl. The term“isoxazolyl” means a heterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) ismethylisoxazolyl, for example —SO₂NHC(O)-3-isoxazolyl-5-methyl or—SO₂NHC(O)-5-isoxazolyl-3-methyl. The term “methylisoxazolyl” means aheterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a)is pyrrolidinyl, for example, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-2-pyrrolidyl, and —SO₂NHC(O)-3-pyrrolidyl. The term“pyrrolidinyl” means a heterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In one particular embodiment, R¹ is —SO₂NHC(O)R^(1a), where R^(1a) ismorpholinyl, for example, —SO₂NHC(O)-4-morpholinyl. The term“morpholinyl” means a heterocyclic compound of the formula:

which is bonded to any available point of attachment and includes:

In yet another particular embodiment, R¹ is —SO₂NHC(O)R^(1a), whereR^(1a) is phenyl optionally substituted with halo. In one embodiment,the phenyl group is unsubstituted and R¹ is —SO₂NHC(O)phenyl. In anotherembodiment, the phenyl group is substituted with 1 or 2 halo atoms. Inyet another embodiment, the halo atoms are fluoro atoms. Examples ofthis embodiment include —SO₂NHC(O)-2-fluorophenyl.

In still another particular embodiment, R¹ is tetrazol-5-yl. In stillanother particular embodiment, R¹ is —C(O)OR^(1b), where R^(1b) is H or—C₁₋₆alkyl, for example, R¹ is —COOH or R¹ is —C(O)OCH₃. In yet anotherembodiment, R¹ is:

And in yet another embodiment, R¹ is:

R³ is selected from —C₂₋₅alkyl and —O—C₁₋₅alkyl. Examples of —C₂₋₅alkylinclude —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —(CH₂)₃CH₃, —CH₂CH(CH₃)₂,—C(CH₃)₃, CH(CH₃)—CH₂CH₃, and —(CH₂)₄CH₃. In one embodiment, R³ ispropyl, ethyl, or butyl. Examples of —O—C₁₋₅alkyl include —OCH₃,—OCH₂CH₃, and —OCH(CH₃)₂. In one embodiment, R³ is ethoxy.

R⁴ is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OR^(4d)), and —CH(R^(4b))C(O)OR^(4c). The R^(4a)moiety is H or —C(O)—C₁₋₆alkyl. The R^(4b) moiety is H or —OH, and theR^(4c) moiety is H or —C₁₋₆alkyl. The R^(4d) moiety is H or—C(O)—R^(4e), and R^(4e) is —C₁₋₆alkyl, —C₁₋₆alkyl-NH₂ or aryl.

In one particular embodiment, R⁴ is —CH₂—SR^(4a). Examples of thisembodiment include —CH₂SH and —CH₂—S—C(O)CH₃.

In another embodiment, R⁴ is —CH₂N(OH)C(O)H. In one particularembodiment, R⁴ is —CH(R^(4b))C(O)NH(OR^(4d)), where R^(4d) moiety is H,such as —CH₂C(O)NH(OH) or —CH(OH)C(O)NH(OH). In another particularembodiment, R⁴ is —CH(R^(4b))C(O)NH(OR^(4d)), where R^(4d) moiety is—C(O)—R^(4e), such as —CH₂C(O)NH—OC(O)CH₃, —CH₂C(O)NH—OC(O)-phenyl, or—CH₂C(O)NH—OC(O)—CH(NH₂)[CH(CH₃)₂].

In one embodiment, R⁴ is —CH(R^(4b))C(O)OR^(4c), where R^(4b) and R^(4c)arc both H, i.e., R⁴ is —CH₂COOH. In another embodiment, R⁴ is—CH(R^(4b))C(O)OR^(4c), where R^(4b) is H, and R^(4c) is —C₁₋₆alkyl.Such examples of R⁴ include —CH₂C(O)OCH₃, —CH₂C(O)OCH₂CH₃,—CH₂C(O)OCH(CH₃)₂, —CH₂C(O)O(CH₂)₂CH₃, —CH₂C(O)O(CH₂)₃CH₃, and—CH₂C(O)O(CH₂)₄CH₃.

In another embodiment, R⁴ is —CH(R^(4b))C(O)OR^(4c), where R^(4b) is H,and R^(4c) is —C₀₋₆alkylenemorpholine, for example R⁴ can be:

In another embodiment, R⁴ is —CH(R^(4b))COOR^(4c), where R^(4b) is H,and R^(4c) is —CH₂OC(O)O—C₁₋₆alkyl or —CH(CH₃)OC(O)O—C₁₋₆alkyl. Examplesof such R⁴ groups include —CH₂C(O)OCH(CH₃)OC(O)OCH₂CH₃ and—CH₂C(O)OCH(CH₃)OC(O)OCH(CH₃)₂. In another embodiment, R⁴ is—CH(R^(4b))COOR^(4c), where R^(4b) is H, and R^(4c) is—CH(CH₃)OC(O)O—C₃₋₇cycloalkyl. Examples of such R⁴ groups include—CH₂C(O)OCH(CH₃)OC(O)O-cyclohexyl. In another embodiment, R⁴ is—CH(R^(4b))C(O)OR^(4c), where R^(4b) is H, and R^(4c) is:

for example R⁴ can be:

In another embodiment, R⁴ is —CH(R^(4b))COOR^(4c), where R^(4b) is —OHand R^(4c) is H or —C₁₋₆alkyl, examples of which include —CH(OH)COOH and—CH(OH)COOCH₃.

R⁵ is selected from —C₁₋₆alkyl, —CH₂-furanyl, —CH₂-thiophenyl, benzyl,and benzyl substituted with one or more halo, —CH₃, or —CF₃ groups. Inone particular embodiment, R⁵ is —C₁₋₆alkyl. Examples of this embodimentinclude i-butyl. In another embodiment, R⁵ is —CH₂-furanyl such as—CH₂-furan-2-yl or —CH₂-furan-3-yl. In one particular embodiment, R⁵ is—CH₂-thiophenyl such as —CH₂-thiophen-2-yl or —CH₂-thiophen-3-yl. In yetanother particular embodiment, R⁵ is benzyl. In still anotherembodiment, R⁵ is benzyl substituted with one or more halo, —CH₃, or—CF₃ groups. Examples of this embodiment include 2-bromobenzyl,2-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,2-methylbenzyl, and 2-trifluoromethylbenzyl.

In one embodiment, R⁴ is —CH(R^(4b))COOR^(4c), where R^(4b) H, and R⁵ isbenzyl substituted with one or more halo, —CH₃, or —CF₃ groups.

In one embodiment of the invention, the compound of formula I is thespecies embodied in formula II:

where Ar, R³, R⁵ and R⁶ are as defined for formula I; or apharmaceutically acceptable salt thereof. In one particular embodiment,Ar is:

R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃, —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl, —COOH, —C(O)OCH₃,

R³ is propyl, ethyl, butyl, or ethoxy;

R⁴ is —CH₂SH, —CH₂—S—C(O)CH₃, —CH₂N(OH)C(O)H, —CH₂C(O)NH(OH),—CH₂C(O)NH—OC(O)CH₃, —CH₂C(O)NH—OC(O)-phenyl,—CH₂C(O)NH—OC(O)—CH(NH₂)[CH(CH₃)₂], —CH(OH)C(O)NH(OH), —CH(OH)COOH,—CH(OH)C(O)OCH₃, —CH₂COOH, —CH₂C(O)OCH₃, —CH₂C(O)OCH₂CH₃,—CH₂C(O)OCH(CH₃)₂, —CH₂C(O)O(CH₂)₂CH₃, —CH₂C(O)O(CH₂)₃CH₃,—CH₂C(O)O(CH₂)₄CH₃, —CH₂C(O)OCH(CH₃)OC(O)OCH₂CH₃,—CH₂C(O)OCH(CH₃)OC(O)OCH(CH₃)₂, —CH₂C(O)OCH(CH₃)OC(O)O-cyclohexyl,

and

R⁵ is i-butyl, —CH₂-furan-2-yl, —CH₂-thiophen-3-yl, benzyl,2-bromobenzyl, 2-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl,4-fluorobenzyl, 2-methylbenzyl, or 2-trifluoromethylbenzyl;

or a pharmaceutically acceptable salt thereof.

In one embodiment, R⁴ is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OR^(4d)), and —CH(R^(4b))C(O)OR^(4c); where R^(4a),R^(4c), and R^(4d) are H; and R^(4b) is as defined for formula I. Inanother aspect, these embodiments have formula II or III.

In yet another embodiment, R⁴ is selected from —CH₂—SR^(4a),—CH(R^(4b))C(O)NH(OR^(4d)), and —CH(R^(4b))C(O)OR^(4c); where R^(4a) is—C(O)—C₁₋₆alkyl; R^(4c) is —C₁₋₆alkyl, —C₀₋₆alkylenemorpholine,—CH₂OC(O)O—C₁₋₆alkyl, —CH(CH₃)OC(O)O—C₁₋₆alkyl,—CH(CH₃)OC(O)O—C₃₋₇cycloalkyl, or:

R^(4d) moiety is —C(O)—R^(4e); and R^(4b) and R^(4e) are as defined forformula I. In one aspect of the invention, these compounds may findparticular utility as prodrugs or as intermediates in the syntheticprocedures described herein. In another aspect, these embodiments haveformula II or III.

In one aspect, the invention relates to compounds of formula IV:

where Ar, R³, R^(4b), R^(4c), and R⁵ arc as defined for formula I; or apharmaceutically acceptable salt thereof. In one particular embodiment,Z is:

Ar is:

R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃, —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl, —COOH, —C(O)OCH₃,

R³ is propyl, ethyl, butyl, or ethoxy;

R^(4b) is H or —OH; and R^(4c) is H, —CH₃, —CH₂CH₃, —CH(CH₃)₂,—(CH₂)₂CH₃, —(CH₂)₃CH₃, —(CH₂)₄CH₃, —CH(CH₃)OC(O)OCH₂CH₃,—CH(CH₃)OC(O)OCH(CH₃)₂, —CH(CH₃)OC(O)O-cyclohexyl,

and

R⁵ is i-butyl, —CH₂-furan-2-yl, —CH₂-thiophen-3-yl, benzyl,2-bromobenzyl, 2-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl,4-fluorobenzyl, 2-methylbenzyl, or 2-trifluoromethylbenzyl;

or a pharmaceutically acceptable salt thereof.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well apharmaceutically acceptable salt thereof.

Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₄alkyl, —C₁₋₅alkyl, —C₂₋₅alkyl, —C₁₋₆alkyl, and —C₁₋₁₀alkyl.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, and the term “—C₃₋₇cycloalkyl” means acycloalkyl group having from 3 to 7 carbon atoms, respectively, wherethe carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₂alkylene-, —C₀₋₃alkylene-, —C₀₋₅alkylene-,—C₀₋₆alkylene-, —C₁₋₂alkylene- and —C₁₋₁₂alkylene-. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like. It is understood that when the alkyleneterm include zero carbons such as —C₀₋₅alkylene- or —C₀₋₆alkylene-, suchterms are intended to include the absence of carbon atoms, that is, thealkylene group is not present except for a covalent bond attaching thegroups separated by the alkylene term.

The term “alkoxy” means a monovalent group of the formula —O-alkyl,where alkyl is as defined herein. Unless otherwise defined, such alkoxygroups typically contain from 1 to 10 carbon atoms and include, forexample, —O—C₁₋₄alkyl and —O—C₁₋₅alkyl. Representative alkoxy groupsinclude, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, sec-butoxy, isobutoxy, t-butoxy and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl and —C₃₋₇cycloalkyl. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like. The term “cycloalkylene” means adivalent aryl group such as —C₄₋₈cycloalkylene.

The term “halo” means fluoro, chloro, bromo and iodo.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used.

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times or 1 to 5 times. For example, a phenyl group that is“optionally substituted” with halo atoms, may be unsubstituted, or itmay contain 1, 2, 3, 4, or 5 halo atoms.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, malcic, oxalic and succinic acids), glucoronic,mandclic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected or blocked from undergoing undesired reactionswith a protecting or blocking group. Functional groups that may beprotected include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like.Representative protecting groups for carboxy groups include esters (suchas a p-methoxybenzyl ester), amides and hydrazides; for amino groups,carbamates (such as t-butoxycarbonyl) and amides; for hydroxyl groups,ethers and esters; for thiol groups, thioethers and thioesters; forcarbonyl groups, acetals and ketals; and the like. Such protectinggroups are well known to those skilled in the art and are described, forexample, in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

As used herein, the term “prodrug” is intended to mean an inactive (orsignificantly less active) precursor of a drug that is converted intoits active form in the body under physiological conditions, for example,by normal metabolic processes. The term is also intended to includecertain protected derivatives of compounds of formula I that may be madeprior to a final deprotection stage. Such compounds may not possesspharmacological activity at AT₁ and/or NEP, but may be administeredorally or parenterally and thereafter metabolized in the body to formcompounds of the invention which are pharmacologically active at AT₁and/or NEP. Thus, all protected derivatives and prodrugs of compoundsformula I are included within the scope of the invention. Prodrugs ofcompounds of formula I having a free carboxyl, sulfhydryl or hydroxygroup can be readily synthesized by techniques that are well known inthe art. These prodrug derivatives are then converted by solvolysis orunder physiological conditions to be the free carboxyl, sulfhydryland/or hydroxy compounds. Exemplary prodrugs include: esters includingC₁₋₆alkylesters and aryl-C₁₋₆alkylesters, carbonate esters, hemi-esters,phosphate esters, nitro esters, sulfate esters, sulfoxides, amides,carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals,ketals, and disulfides. In one embodiment, the compounds of formula Ihave a free sulfhydryl or a free carboxyl and the prodrug is an esterderivative thereof, i.e., the prodrug is a thioester such as —SC(O)CH₃or an ester such as —C(O)OCH₃.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, for example, a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate or prevent the symptoms of hypertension, or to treatthe underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessary be atherapeutic result. For example, when studying a system comprising anAT₁ receptor, an “effective amount” may be the amount needed toantagonize the receptor.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, that is, by prophylactic treatment of a patient; (b)ameliorating the disease or medical condition such as by eliminating orcausing regression of the disease or medical condition in a patient; (c)suppressing the disease or medical condition such as by slowing orarresting the development of the disease or medical condition in apatient; or (d) alleviating the symptoms of the disease or medicalcondition in a patient. For example, the term “treating hypertension”would include preventing hypertension from occurring, amelioratinghypertension, suppressing hypertension, and alleviating the symptoms ofhypertension (for example, lowering blood pressure). The term “patient”is intended to include those mammals, such as humans, that are in needof treatment or disease prevention or that are presently being treatedfor disease prevention or treatment of a specific disease or medicalcondition, as well as test subjects in which compounds of the inventionarc being evaluated or being used in a assay, for example an animalmodel.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. While optimum reaction conditions will typically varydepending on various reaction parameters such as the particularreactants, solvents and quantities used, those of ordinary skill in theart can readily determine suitable reaction conditions using routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, arc described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, New York, 1999, and references cited therein. Morespecifically, the following abbreviations and reagents are used in theschemes presented below:

P¹ represents an “amino-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at an aminogroup. Representative amino-protecting groups include, but are notlimited to, t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl(Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), and the like. Standard deprotectiontechniques are used to remove the P¹ group. For example, a BOC group canbe removed using an acidic reagent such as TFA in DCM or HCl in1,4-dioxane, while a Cbz group can be removed by employing catalytichydrogenation conditions such as H₂ (1 atm) and 10% Pd/C in an alcoholicsolvent (“H₂/Pd/C”).

P² represents a “carboxy-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at acarboxy group. Representative carboxy-protecting groups include, but arenot limited to, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl(PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), diphenylmethyl (benzhydryl, DPM) and thelike. Standard deprotection techniques and reagents are used to removethe P² group, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when P² is methyl,an acid such as TFA or HCl is commonly used when P² is t-butyl, andH₂/Pd/C may be used when P² is benzyl.

P³ represents a “thiol-protecting group,” a term used herein to mean aprotecting group suitable for preventing undesired reactions at a thiolgroup. Representative thiol-protecting groups include, but are notlimited to, ethers, esters such as —C(O)CH₃, and the like. Standarddeprotection techniques and reagents such as NaOH, primary alkylamines,and hydrazine, may be used to remove the P³ group.

P⁴ represents a “tetrazole-protecting group,” a term used herein to meana protecting group suitable for preventing undesired reactions at atetrazole group. Representative tetrazole-protecting groups include, butare not limited to trityl and diphenylmethyl. Standard deprotectiontechniques and reagents such as TFA in DCM or HCl in 1,4-dioxane may beused to remove the P⁴ group.

P⁵ represents a “hydroxyl-protecting group,” a term that is used hereinto mean a protecting group suitable for preventing undesired reactionsat a hydroxyl group. Representative hydroxyl-protecting groups include,but are not limited to C₁₋₆alkyls, silyl groups includingtriC₁₋₆alkylsilyl groups, such as trimethylsilyl (TMS), triethylsilyl(TES), and tert-butyldimethylsilyl (TBDMS); esters (acyl groups)including C₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, andaromatic acyl groups such as benzoyl; arylmethyl groups such as benzyl(Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); and the like. Standard deprotection techniques andreagents are used to remove the P⁵ group, and may vary depending uponwhich group is used. For example, H₂/Pd/C is commonly used when P⁵ isbenzyl, while NaOH is commonly used when P⁵ is an acyl group.

P⁶ represents a “sulfonamide-protecting group,” a term that is usedherein to mean a protecting group suitable for preventing undesiredreactions at a sulfonamide group. Representative sulfonamide-protectinggroups include, but are not limited to t-butyl and acyl groups.Exemplary acyl groups include aliphatic lower acyl groups such as theformyl, acetyl, phenylacetyl, butyryl, isobutyryl, valeryl, isovaleryland pivaloyl groups, and aromatic acyl groups such as the benzoyl and4-acetoxybenzoyl. Standard deprotection techniques and reagents are usedto remove the P⁶ group, and may vary depending upon which group is used.For example, HCl is commonly used when P⁶ is t-butyl, while NaOH iscommonly used when P⁶ is an acyl group.

In addition, L is used to designate a “leaving group,” a term usedherein to mean a functional group or atom which can be displaced byanother functional group or atom in a substitution reaction, such as anucleophilic substitution reaction. By way of example, representativeleaving groups include chloro, bromo and iodo groups; sulfonic estergroups, such as mesylate, triflate, tosylate, brosylate, nosylate andthe like; and acyloxy groups, such as acetoxy, trifluoroacetoxy and thelike.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N,N-diisopropylethylamine (DIPEA), sodium hydroxide, potassiumhydroxide, potassium t-butoxide, and metal hydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), dimethyl sulfoxide(DMSO), toluene, dichloromethane (DCM), chloroform (CHCl₃), carbontetrachloride (CCl₄), 1,4-dioxane, methanol, ethanol, water, and thelike.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC),carbonyldiimidazole (CDI), and the like. Coupling reactions areconducted in an inert diluent in the presence of a base such as DIPEA,and are performed under conventional amide bond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78° C. to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying (forexample, over MgSO₄, over Na₂SO₄, or in vacuo); filtering; crystallizing(for example, from EtOAc and hexane); being concentrated (for example,in vacuo); and/or purification (e.g., silica gel chromatography, flashchromatography, preparative HPLC, reverse phase-HPLC, orcrystallization).

By way of illustration, compounds of formula I, as well as their salts,solvates, and prodrugs can be prepared by coupling a compound of formula1 with a compound of formula 2:

Ar* represents Ar—R¹*, where R¹* is R¹ or a protected form of R¹, forexample, -tetrazolyl-BOC or a precursor of R¹ such as —CN that is thenconverted to tetrazolyl. R⁴* represents R⁴ or a protected form of R⁴.Therefore, when R¹* represents R¹ and R⁴* represents R⁴, the reaction iscomplete after the coupling step.

On the other hand, when R¹* represents a protected form of R¹ and/or R⁴*represents a protected form of R⁴, a subsequent global or sequentialdeprotection step would yield the non-protected compound. Similarly,when R¹* represents a precursor of R¹, a subsequent conversion stepwould yield the desired compound. Reagents and conditions for thedeprotection vary with the nature of protecting groups in the compound.Thus, one method of preparing compounds of the invention involvescoupling compounds (1) and (2), with an optional deprotection step whenR¹* is a protected form of R¹ and/or R⁴* is a protected form of R⁴, thusforming a compound of formula I or a pharmaceutically acceptable saltthereof.

Certain intermediates described herein are believed to be novel andaccordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formulas V, VI andVII, or a salt thereof:

where Ar* is Ar—R¹*; Ar, Z, R³, R⁴, and R⁵ are as defined for formula I;and R¹* is —SO₂NH—P⁶ or tetrazolyl-P⁴; where P⁴ is atetrazole-protecting group and P⁶ is a sulfonamide-protecting group;

where Ar, Z, R³, and R⁵ are as defined for formula I; R⁴* is —CH₂—S—P³,—CH₂—N(O—P⁵)—C(O)H, —CH(R^(4b))C(O)NH(O—P⁵), or —CH(R^(4b))C(O)O—P²; andR^(4b) is as defined for formula I; where P² is a carboxy-protectinggroup, P³ is a thiol-protecting group, P⁵ is a hydroxyl-protectinggroup; and

where Ar* is Ar—R¹*; Ar, Z, R³, and R⁵ are as defined for formula I; R¹*is —SO₂NH—P⁶ or tetrazolyl-P⁴; R⁴* is —CH₂—S—P³, —CH₂—N(O—P⁵)—C(O)H,—CH(R^(4b))C(O)NH(O—P⁵), or —CH(R^(4b))C(O)O—P²; and R^(4b) is asdefined for formula I; where P² is a carboxy-protecting group, P³ is athiol-protecting group, P⁴ is a tetrazole-protecting group, P⁵ is ahydroxyl-protecting group, and P⁶ is a sulfonamide-protecting group.Thus, another method of preparing compounds of the invention involvesdeprotecting a compound of formula V, VI, or VII.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess angiotensin II type 1 (AT₁) receptorantagonist activity. In one embodiment, compounds of the invention areselective for inhibition of the AT₁ receptor over the AT₂ receptor.Compounds of the invention also possess neprilysin (NEP) inhibitionactivity, that is, the compounds are able to inhibit enzyme-substrateactivity. In another embodiment, the compounds do not exhibitsignificant inhibitory activity of the angiotensin-converting enzyme.Compounds of formula I may be active drugs as well as prodrugs. Thus,when discussing the activity of compounds of the invention, it isunderstood that any such prodrugs have the expected activity oncemetabolized.

One measure of the affinity of a compound for the AT₁ receptor is theinhibitory constant (K_(i)) for binding to the AT₁ receptor. The pK_(i)value is the negative logarithm to base 10 of the K_(i). One measure ofthe ability of a compound to inhibit NEP activity is the inhibitoryconcentration (IC₅₀), which is the concentration of compound thatresults in half-maximal inhibition of substrate conversion by the NEPenzyme. The pIC₅₀ value is the negative logarithm to base 10 of theIC₅₀. Compounds of the invention that have both AT₁receptor-antagonizing activity and NEP enzyme-inhibiting activity are ofparticular interest, including those that exhibit a pK_(i) at the AT₁receptor greater than or equal to about 5.0, and exhibit a pIC₅₀ for NEPgreater than or equal to about 5.0.

In one embodiment, compounds of interest have a pK_(i) at the AT₁receptor≧about 6.0, a pK_(i) at the AT₁ receptor≧about 7.0, or a pK_(i)at the AT₁ receptor≧about 8.0. Compounds of interest also include thosehaving a pIC₅₀ for NEP≧about 6.0 or a pIC₅₀ for NEP≧about 7.0. Inanother embodiment, compounds of interest have a pK_(i) at the AT₁receptor within the range of about 8.0-10.0 and a pIC₅₀ for NEP withinthe range of about 7.0-10.0.

In another embodiment, compounds of particular interest have a pK_(i)for binding to an AT₁ receptor greater than or equal to about 7.5 and aNEP enzyme pIC₅₀ greater than or equal to about 7.0. In anotherembodiment, compounds of interest have a pK_(i) greater than or equal toabout 8.0 and a pIC₅₀ greater than or equal to about 8.0.

It is noted that in some cases, compounds of the invention, while stillhaving dual activity, may possess either weak AT₁ receptor antagonistactivity or weak NEP inhibition activity. In such cases, those of skillin the art will recognize that these compounds still have utility asprimarily either a NEP inhibitor or a AT₁ receptor antagonist,respectively, or have utility as research tools.

Exemplary assays to determine properties of compounds of the invention,such as the AT₁ receptor binding and/or NEP inhibiting activity, aredescribed in the Examples and include by way of illustration and notlimitation, assays that measure AT₁ and AT₂ binding and NEP inhibition(described in Assay 1). Useful secondary assays include assays tomeasure ACE inhibition (also described in Assay 1) and aminopeptidase P(APP) inhibition (described in Sulpizio et al. (2005) JPET315:1306-1313). A pharmacodynamic assay to assess the in vivo inhibitorypotencies for ACE, AT₁, and NEP in anesthetized rats is described inAssay 2 (see also Seymour et al. (1985) Hypertension 7(SupplI):I-35-I-42 and Wigle et al. (1992) Can. J. Physiol. Pharmacol.70:1525-1528), where AT₁ inhibition is measured as the percentinhibition of the angiotensin II pressor response, ACE inhibition ismeasured as the percent inhibition of the angiotensin I pressorresponse, and NEP inhibition is measured as increased urinary cyclicguanosine 3′,5′-monophosphate (cGMP) output. Useful in vivo assaysinclude the conscious spontaneously hypertensive rat (SHR) model, whichis a renin dependent hypertension model that is useful for measuring AT₁receptor blocking (described in Assay 3; see also Intengan et al. (1999)Circulation 100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362), and the conscious desoxycorticosteroneacetate-salt (DOCA-salt) rat model, which is a volume dependenthypertension model that is useful for measuring NEP activity (describedin Assay 4; see also Trapani et al. (1989) J. Cardiovasc. Pharmacol.14:419-424, Intengan et al. (1999) Hypertension 34(4):907-913, andBadyal et al. (2003) supra). Both the SHR and DOCA-salt models areuseful for evaluating the ability of a test compound to reduce bloodpressure. The DOCA-salt model is also useful to measure a testcompound's ability to prevent or delay a rise in blood pressure.Compounds of the invention are expected to antagonize the AT₁ receptorand/or inhibit the NEP enzyme in any of the assays listed above, orassays of a similar nature. Thus, the aforementioned assays are usefulin determining the therapeutic utility of compounds of the invention,for example, their utility as antihypertensive agents. Other propertiesand utilities of compounds of the invention can be demonstrated usingother in vitro and in vivo assays well-known to those skilled in theart.

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions responsive to AT₁ receptorantagonism and/or NEP inhibition. Thus it is expected that patientssuffering from a disease or disorder that is treated by antagonizing theAT₁ receptor and/or by inhibiting the NEP enzyme can be treated byadministering a therapeutically effective amount of a compound of theinvention. For example, by antagonizing the AT₁ receptor and thusinterfering with the action of angiotensin II on its receptors, thesecompounds are expected to find utility in preventing the increase inblood pressure produced by angiotensin II, a potent vasopressor. Inaddition, by inhibiting NEP, the compounds are also expected topotentiate the biological effects of endogenous peptides that aremetabolized by NEP, such as the natriuretic peptides, bombesin,bradykinins, calcitonin, endothelins, enkephalins, neurotensin,substance P and vasoactive intestinal peptide. For example, bypotentiating the effects of the natriuretic peptides, compounds of theinvention are expected to be useful to treat glaucoma. These compoundsare also expected to have other physiological actions, for example, onthe renal, central nervous, reproductive and gastrointestinal systems.

Compounds of the invention are expected to find utility in treatingand/or preventing medical conditions such as cardiovascular and renaldiseases. Cardiovascular diseases of particular interest include heartfailure such as congestive heart failure, acute heart failure, chronicheart failure, and acute and chronic decompensated heart failure. Renaldiseases of particular interest include diabetic nephropathy and chronickidney disease. One embodiment of the invention relates to a method fortreating hypertension, comprising administering to a patient atherapeutically effective amount of a compound of the invention.Typically, the therapeutically effective amount is the amount that issufficient to lower the patient's blood pressure. In one embodiment, thecompound is administered as an oral dosage form.

Another embodiment of the invention relates to a method for treatingheart failure, comprising administering to a patient a therapeuticallyeffective amount of a compound of the invention. Typically, thetherapeutically effective amount is the amount that is sufficient tolower blood pressure and/or improve renal functions. In one embodiment,the compound is administered as an intravenous dosage form. When used totreat heart failure, the compound may be administered in combinationwith other therapeutic agents such as diuretics, natriuretic peptides,and adenosine receptor antagonists.

Compounds of the invention are also expected to be useful inpreventative therapy, for example in preventing the progression ofcardiac insufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

In addition, as NEP inhibitors, compounds of the invention are expectedto inhibit enkephalinase, which will inhibit the degradation ofendogenous enkephalins and thus such compounds may also find utility asanalgesics. Due to their NEP inhibition properties, compounds of theinvention are also expected to be useful as antitussive agents andantidiarrheal agents (for example, for the treatment of waterydiarrhea), as well as find utility in the treatment of menstrualdisorders, preterm labor, pre-eclampsia, endometriosis, reproductivedisorders (for example, male and female infertility, polycystic ovariansyndrome, implantation failure), and male and female sexual dysfunction,including male erectile dysfunction and female sexual arousal disorder.More specifically, the compounds of the invention are expected to beuseful in treating female sexual dysfunction, which is often defined asa female patient's difficulty or inability to find satisfaction insexual expression. This covers a variety of diverse female sexualdisorders including, by way of illustration and not limitation,hypoactive sexual desire disorder, sexual arousal disorder, orgasmicdisorder and sexual pain disorder. When used to treat such disorders,especially female sexual dysfunction, compounds of the invention may becombined with one or more of the following secondary agents: PDE5inhibitors, dopamine agonists, estrogen receptor agonists and/orantagonists, androgens, and estrogens.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Since compounds of the invention possess AT₁ receptor antagonistactivity and/or NEP enzyme inhibition activity, such compounds are alsouseful as research tools for investigating or studying biologicalsystems or samples having AT₁ receptors or a NEP enzyme, for example tostudy diseases where the AT₁ receptor or NEP enzyme plays a role. Anysuitable biological system or sample having AT₁ receptors and/or a NEPenzyme may be employed in such studies which may be conducted either invitro or in vivo. Representative biological systems or samples suitablefor such studies include, but are not limited to, cells, cellularextracts, plasma membranes, tissue samples, isolated organs, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, humans, and soforth), and the like, with mammals being of particular interest. In oneparticular embodiment of the invention an AT₁ receptor in a mammal isantagonized by administering an AT₁-antagonizing amount of a compound ofthe invention. In another particular embodiment, NEP enzyme activity ina mammal is inhibited by administering a NEP-inhibiting amount of acompound of the invention. Compounds of the invention can also be usedas research tools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprisingan AT₁ receptor and/or a NEP enzyme is typically contacted with an AT₁receptor-antagonizing or NEP enzyme-inhibiting amount of a compound ofthe invention. After the biological system or sample is exposed to thecompound, the effects of antagonizing the AT₁ receptor and/or inhibitingthe NEP enzyme are determined using conventional procedures andequipment, such as by measuring receptor binding in a binding assay ormeasuring ligand-mediated changes in a functional assay. Exposureencompasses contacting cells or tissue with the compound, administeringthe compound to a mammal, for example by i.p., i.v. or s.c.administration, and so forth. This determining step can involvemeasuring a response (a quantitative analysis) or can involve making anobservation (a qualitative analysis). Measuring a response involves, forexample, determining the effects of the compound on the biologicalsystem or sample using conventional procedures and equipment, such asradioligand binding assays and measuring ligand-mediated changes infunctional assays. The assay results can be used to determine theactivity level as well as the amount of compound necessary to achievethe desired result, that is, an AT₁ receptor-antagonizing and/or a NEPenzyme-inhibiting amount. Typically, the determining step will involvedetermining the AT₁ receptor ligand-mediated effects and/or determiningthe effects of inhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds having AT₁receptor-antagonizing activity and/or NEP-inhibiting activity. In thismanner, a compound of the invention is used as a standard in an assay toallow comparison of the results obtained with a test compound and withcompounds of the invention to identify those test compounds that haveabout equal or superior activity, if any. For example, K_(i) data (asdetermined, for example, by a binding assay) for a test compound or agroup of test compounds is compared to the K_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a K_(i) value about equalor superior to a compound of the invention, if any. This aspect of theinvention includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include an AT₁ receptorbinding assay and a NEP enzyme inhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent, ” to distinguish it from other componentsof the formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt%, withthe actual amount depending upon the formulation itself, the route ofadministration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In formulations where the compound of the invention contains a thiolgroup, additional consideration may be given to minimize or eliminateoxidation of the thiol to form a disulfide. In solid formulations, thismay be accomplished by reducing the drying time, decreasing the moisturecontent of the formulation, and including materials such as ascorbicacid, sodium ascorbate, sodium sulfite and sodium bisulfate, as well asmaterials such as a mixture of lactose and microcrystalline cellulose.In liquid formulations, stability of the thiol may be improved by theaddition of amino acids, antioxidants, or a combination of disodiumedetate and ascorbic acid.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention arc suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages, or dimerization ofthiols that may be present in the compound. These formulations may berendered sterile by use of a sterile injectable medium, a sterilizingagent, filtration, irradiation, or heat. In one particular embodiment,the parenteral formulation comprises an aqueous cyclodextrin solution asthe pharmaceutically acceptable carrier. Suitable cyclodextrins includecyclic molecules containing six or more α-D-glucopyranose units linkedat the 1,4 positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl etherβ-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the compounds of the invention may be administered incombination with one or more other therapeutic agents. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”) selected from the group ofdiuretics, β₁ adrenergic receptor blockers, calcium channel blockers,angiotensin-converting enzyme inhibitors, AT₁ receptor antagonists,neprilysin inhibitors, non-steroidal anti-inflammatory agents,prostaglandins, anti-lipid agents, anti-diabetic agents, anti-thromboticagents, renin inhibitors, endothelin receptor antagonists, endothelinconverting enzyme inhibitors, aldosterone antagonists,angiotensin-converting enzyme/neprilysin inhibitors, and combinationsthereof. Such therapeutic agents are well known in the art, and specificexamples are described herein. By combining a compound of the inventionwith a secondary agent, triple therapy can be achieved; AT₁ receptorantagonist activity, NEP inhibition activity, and activity associatedwith the secondary agent (for example, β₁ adrenergic receptor blocker)can be achieved using only two active components. Since compositionscontaining two active components are typically easier to formulate thancompositions containing three active components, such two-componentcompositions provide a significant advantage over compositionscontaining three active components. Accordingly, in yet another aspectof the invention, a pharmaceutical composition comprises a compound ofthe invention, a second active agent, and a pharmaceutically acceptablecarrier. Third, fourth etc. active agents may also be included in thecomposition. In combination therapy, the amount of compound of theinvention that is administered, as well as the amount of secondaryagents, may be less than the amount typically administered inmonotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc,) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, a compound of the invention is administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosernide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone, chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone,flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide,methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide,quinethazone, teclothiazide, and trichloromethiazide; and combinationsthereof. In a particular embodiment, the diuretic is selected fromamiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide,ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, metolazone, torsemide, triamterene, andcombinations thereof. The diuretic will be administered in an amountsufficient to provide from about 5-50 mg per day, more typically 6-25 mgper day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination witha β₁ adrenergic receptor blocker. Representative β₁ adrenergic receptorblockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁ adrenergic receptor blocker is selected fromatenolol, bisoprolol, metoprolol, propranolol, sotalol, and combinationsthereof.

In one embodiment, a compound of the invention is administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof.

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril, moveltopril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof. In aparticular embodiment, the ACE inhibitor is selected from: benazepril,enalapril, lisinopril, ramipril, and combinations thereof.

In one embodiment, a compound of the invention is administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, benzyllosartan, candesartan, candesartancilexetil, elisartan, embusartan, enoltasosartan, eprosartan, fonsartan,forasartan, glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan, opomisartan, pratosartan, ripisartan,saprisartan, saralasin, sarmesin, tasosartan, telmisartan, valsartan,zolasartan, and combinations thereof. In a particular embodiment, theARB is selected from candesartan, eprosartan, irbesartan, losartan,olmesartan, irbesartan, saprisartan, tasosartan, telmisartan, andcombinations thereof. Exemplary salts include eprosartan mesylate,losartan potassium salt, and olmesartan medoxomil. Typically, the ARBwill be administered in an amount sufficient to provide from about 4-600mg per dose, with exemplary daily dosages ranging from 20-320 mg perday.

In another embodiment, a compound of the invention is administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: candoxatril; candoxatrilat;dexecadotril ((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycinebenzyl ester); CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155 (N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R) -hydroxy-L-proline benzyl ester);3-(1-carbamoylcyclohexyl)propionic acid derivatives described in WO2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-β-phenylpropyl]-(-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racccadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (AHU-377 acid parent and AHU-377 ester prodrug; WO2007/056546); daglutril[(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from candoxatril, candoxatrilat, CGS-24128, phosphoramidon,SCH-32615, SCH-34826, SQ-28603, thiorphan, AHU-377 (parent or prodrug),and combinations thereof. The NEP inhibitor will be administered in anamount sufficient to provide from about 20-800 mg per day, with typicaldaily dosages ranging from 50-700 mg per day, more commonly 100-600 or100-300 mg per day.

In yet another embodiment, a compound of the invention is administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,amoxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but arc not limited to, statins such as atorvastatin,fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin;cholesteryl ester transfer proteins (CETPs); and combinations thereof.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; meglitinides such as repaglinidc;oxadiazolidinediones; sulfonylurcas such as chlorpropamidc, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In one embodiment, a compound of the invention is administered incombination with an anti-thrombotic agent. Representativeanti-thrombotic agents include, but are not limited to, aspirin,anti-platelet agents, heparin, and combinations thereof. Compounds ofthe invention may also be administered in combination with a renininhibitor, examples of which include, but are not limited to, aliskiren,enalkiren, remikiren, and combinations thereof. In another embodiment, acompound of the invention is administered in combination with anendothelin receptor antagonist, representative examples of whichinclude, but are not limited to, bosentan, darusentan, tezosentan, andcombinations thereof. Compounds of the invention may also beadministered in combination with an endothelin converting enzymeinhibitor, examples of which include, but are not limited to,phosphoramidon, CGS 26303, and combinations thereof. In yet anotherembodiment, a compound of the invention is administered in combinationwith an aldosterone antagonist, representative examples of whichinclude, but are not limited to, eplerenone, spironolactone, andcombinations thereof.

Combined therapeutic agents may also be helpful in further combinationtherapy with compounds of the invention. For example, a combination ofthe ACE inhibitor enalapril (in the maleate salt form) and the diuretichydrochlorothiazide, which is sold under the mark Vaseretic®, or acombination of the calcium channel blocker amlodipine (in the besylatesalt form) and the ARB olmesartan (in the medoxomil prodrug form), or acombination of a calcium channel blocker and a statin, all may also beused with the compounds of the invention. Dual-acting agents may also behelpful in combination therapy with compounds of the invention. Forexample, angiotensin-converting enzyme/neprilysin (ACE/NEP) inhibitorssuch as: AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]aceticacid); CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gcmpatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105(N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat;SA-898((2R,4R)-N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACE/NEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof.

Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine. Exemplary vasopressin receptorantagonists include tolvaptan.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (300 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 260 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum^( ®) K(magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning:

-   -   ACE angiotensin converting enzyme    -   AcOH acetic acid    -   APP aminopeptidase P    -   AT₁ angiotensin II type 1 (receptor)    -   AT₂ angiotensin II type 2 (receptor)    -   BSA bovine serum albumin    -   DCM dichloromethane or methylene chloride    -   DIPEA N,N-diisopropylethylamine    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   Dnp 2,4-dinitrophenyl    -   DOCA deoxycorticosterone acetate    -   EDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide        hydrochloride    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   EtOH ethanol    -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hcxafluorophosphatc    -   HOAt 1-hydroxy-7-azabenzotriazole    -   Mca (7-methoxycoumarin-4-yl)acyl    -   MeCN acetonitrile    -   MeOH methanol    -   NBS N-bromosuccinimide    -   NEP neprilysin (EC 3.4.24.11)    -   PBS phosphate buffered saline    -   SHR spontaneously hypertensive rat    -   TFA trifluoroacctic acid    -   THF tetrahydrofuran    -   Tris tris(hydroxymethyl)aminomethane    -   Tween-20 polyethylene glycol sorbitan monolaurate

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haën, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN /1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 15-Propyl-1-[2′-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicAcid

Ethyl 3-n-propylpyrazole-5-carboxylate (1.0 g, 5.5 mmol),5-(4′-bromomethylbiphenyl-2-yl)-1-trityl-1H-tetrazole (3.1 g, 5.5 mmol),and potassium carbonate (1.5 g, 11.0 mmol) were dissolved in DMF (40.0mL, 516 mmol) and the mixture was stirred at room temperature overnight.LCMS showed the reaction was nearly complete, with a 1:1 mixture ofregioisomers. The mixture was partially concentrated, then partitionedbetween water and EtOAc. The EtOAc layer was then dried over Na₂SO₄ andconcentrated. The regioisomers were separated by flash chromatography(0-50% EtOAc/hexanes, dry load). Regioisomer identities were confirmedby NOE:5-propyl-1-[2′-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ethyl ester (1.2 g) and5-propyl-2-[2′-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-2H-pyrazole-3-carboxylicacid ethyl ester (1.1 g).

5-Propyl-1-[2′-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ethyl ester (400 mg, 607 μmol) was dissolved in THF (12.0 mL, 148mmol). A solution of LiOH monohydrate (127 mg, 3.04 mmol) in water (4.0mL, 222 mmol) was then added and the mixture was stirred at roomtemperature for about 19 hours. The mixture was then heated to 60° C.overnight. LCMS showed that the reaction was complete, but that twoadditional side products had also formed. The mixture was partitionedbetween EtOAc and a saturated ammonium chloride solution. The EtOAclayer was dried over Na₂SO₄ and concentrated to yield the title compound(340 mg). LCMS showed that the side products were no longer present.

Example 1(2R,3R)-3-(1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicAcid

A solution of5-propyl-1-[2′-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid (475 mg, 753 mmol), HATU (286.3 mg, 753.1 μmol), DIPEA (787.0 μL,4518 μmol), and DMF (21.3 mL, 274.7 mmol) was stirred to pre-activatethe acid. (2R,3R)-3-Amino-2-hydroxy-4-phenylbutyric acid.HCl (175 mg,755 μmol) was added and the resulting mixture was heated at 45° C. andstirred overnight. The mixture was concentrated to yield the protectedintermediate, (2R,3R)-2-hydroxy-4-phenyl-3-({5-propyl-1-[2′-(1-trityl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid as a residue, which was used directly in the next step. The residuewas dissolved in 1,4-dioxane (10 mL, 100 mmol) and 4.0 M of HCl in1,4-dioxane (10 mL, 40 mmol) was added. The resulting solution wasstirred for 90 minutes. The mixture was concentrated, re-dissolved inwater/MeCN/TFA and purified using reverse phase liquid chromatography toyield the title compound (176 mg; 96% purity). MS m/z: [M+H]⁺ calcd forC₃₁H₃₁N₇O₄, 566.24; found 566.6.

Preparation 2 (Tetrazol-5-yl)phenylboronic Acid

[2-(1-Trityltetrazol-5-yl)phenyl]boronic acid (11.5 g, 26.6 mmol) wascombined with 1,4-dioxane (41.5 mL, 532.1 mmol) and 4 M of HCl in1,4-dioxane (13.3 mL, 53.2 mmol). The mixture was stirred for 2 hours.EtOAc (100 mL) was added. 10 M NaOH was added until pH˜9, with constantstirring. The organic layer was extracted and discarded. The aqueouslayer was acidified to pH˜2 with DCM (10 mL). The product crashed outand was filtered and dried to obtain the title compound (3.5 g) as awhite solid.

Preparation 3 1-(4-Bromobenzyl)-5-propyl-1H-pyrazole-3-carboxylic AcidEthyl Ester

Ethyl 3-n-propylpyrazole-5-carboxylate (8.0 g, 43.0 mmol), 4-bromobenzylbromide (11.0 g, 43.0 mmol) and potassium carbonate (5.9 g, 43.0 mmol)were dissolved in DMF (433 mL, 5590 mmol). The resulting suspension wasstirred at room temperature over approximately 48 hours. The materialwas concentrated, then diluted with aqueous NaHCO₃ and diethyl ether.The aqueous layer was further extracted with diethyl ether. The combinedorganics were dried over MgSO₄, filtered and concentrated. The residuewas purified using flash chromatography (1^(st) 40:1 hexanes/EtOAc;2^(nd) 5:1 hexanes/EtOAc). The two isomers were separated and analyzedby NMR and NOE: 1-(4-bromobenzyl)-5-propyl-1H-pyrazole-3-carboxylic acidethyl ester (5.7 g) and2-(4-bromobenzyl)-5-propyl-2H-pyrazole-3-carboxylic acid ethyl ester.

Preparation 45-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicAcid

A solution of (tetrazol-5-yl)phenylboronic acid (650 mg, 3.4 mmol),1-(4-bromobenzyl)-5-propyl-1H-pyrazole-3-carboxylic acid ethyl ester(1.0 g, 2.9 mmol), tetrakis(triphenylphosphine)palladium(0) (200 mg, 0.1mmol), 1.0 M NaOH in water (9.0 mL, 9.0 mmol) and MeOH (14 mL, 350 mmol)was sparged shortly, capped under nitrogen and heated in the microwaveat 90° C. for 2 hours. The mixture was concentrated to remove the MeOH,then extracted with EtOAc. The organic layer was extracted with 1 NNaOH. The combined aqueous layers were acidified with 1 N HCl to pH 3-4and extracted with EtOAc. The organics were dried over MgSO₄, filteredand concentrated. The reaction was repeated twice due to the volumerestriction of the microwave reaction vessel, and the residues werecombined to yield the title compound (2.2 g), which was used withoutfurther purification.

Example 2(R)-4-Phenyl-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricAcid

A solution of5-propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid (200 mg, 510 μmol), HOAt (77.1 mg, 566.4 μmol), EDCI (108.6 mg,566.4 μmol), and 2,6-lutidine (357.8 μL, 3.1 mmol) in DCM (6.1 mL, 94.7mmol) was stirred to pre-activate the acid. After 30 minutes, themixture was cooled at 0° C. and (R)-3-amino-4-phenylbutyric acidhydrochloride (122.2 mg,566.4 μmol) was added. The resulting solutionwas slowly warmed to room temperature and stirred overnight. The mixturewas then concentrated, re-dissolved in water/MeCN/TFA and purified usingreverse phase liquid chromatography to yield the title compound (59 mg;92% purity). MS m/z: [M+H]⁺ calcd for C₃₁H₃₁N₇O₃, 550.25; found 550.4.

Example 3

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds3-1 to 3-38, having the following formula, were prepared:

MS m/z: [M + H]⁺ # R R³ R⁴ R⁵ Formula calcd found 1 3-F ethoxy —CH₂SHbenzyl C₂₉H₂₈FN₇O₂S 558.20 558.6 2 3-F propyl —CH₂SH benzyl C₃₀H₃₀FN₇OS556.22 556.4 3 3-F propyl —CH₂SH i-butyl C₂₇H₃₂FN₇OS 522.24 522.6 4 —ethoxy —CH₂SH benzyl C₂₉H₂₉N₇O₂S 540.21 540.6 5 — ethoxy —CH₂SH i-butylC₂₆H₃₁N₇O₂S 506.23 506.4 6 — propyl —CH₂SH benzyl C₃₀H₃₁N₇OS 538.23538.8 7 — propyl —CH₂SH i-butyl C₂₇H₃₃N₇OS 504.25 504.6 8 3-F ethoxy—CH(OH)—COOH benzyl C₃₀H₂₈FN₇O₅ 586.21 586.4 9 — ethoxy —CH(OH)—COOHbenzyl C₃₀H₂₉N₇O₅ 568.22 568.4 10 2-F propyl —CH(OH)—COOH benzylC₃₁H₃₀FN₇O₄ 584.23 584.4 11 2-F propyl —CH₂SH benzyl C₃₀H₃₀FN₇OS 556.22556.4 12 — propyl —CH(OH)—C(O)OCH₃ benzyl C₃₂H₃₃N₇O₄ 580.26 580.4 13 —propyl —CH₂COOH 2-Br- C₃₁H₃₀BrN₇O₃ 628.16 628.4 benzyl 14 — propyl—CH₂—N(OH)—C(O)H benzyl C₃₁H₃₂N₈O₃ 565.26 565.4 15 — propyl—CH₂—C(O)NH(OH) 2-Cl- C₃₁H₃₁ClN₈O₃ 599.22 599.6 benzyl 16 — propyl—CH₂—C(O)NH(OH) benzyl C₃₁H₃₂N₈O₃ 565.26 565.6 17 — propyl—CH(OH)—C(O)NH(OH) benzyl C₃₁H₃₂N₈O₄ 581.25 581.4 18 2-F propyl —CH₂COOH2-Cl- C₃₁H₂₉ClFN₇O₃ 602.20 602.6 benzyl 19 2-F propyl —CH₂COOH 2-CF₃-C₃₂H₂₉F₄N₇O₃ 636.23 636.4 benzyl 20 3-F propyl —CH₂COOH 2-Cl-C₃₁H₂₉ClFN₇O₃ 602.20 602.6 benzyl 21 3-F propyl —CH₂COOH 2-CF₃-C₃₂H₂₉F₄N₇O₃ 636.23 636.2 benzyl 22 — propyl —CH(OH)—COOH 2-Cl-C₃₁H₃₀ClN₇O₄ 600.21 600.4 benzyl 23 3-F propyl —CH(OH)—COOH benzylC₃₁H₃₀FN₇O₄ 584.23 584.4 24 2-F propyl —CH(OH)—COOH benzyl C₃₁H₃₀FN₇O₄584.23 584.4 25 — propyl —CH₂—N(OH)—C(O)H benzyl C₂₈H₃₄N₈O₃ 531.28 531.626 3,5- propyl —CH(OH)—COOH benzyl C₃₁H₂₉F₂N₇O₄ 602.23 602.6 diF 27 —propyl —CH₂—C(O)OCH₃ benzyl C₃₂H₃₃N₇O₃ 564.26 564.6 28 — propyl—CH₂—C(O)OCH₃ 2-Cl- C₃₂H₃₂ClN₇O₃ 598.23 598.4 benzyl 29 — propyl—CH₂—C(O)OCH₃ 2-CH₃- C₃₃H₃₅N₇O₃ 578.28 578.6 benzyl 30 3-F propyl—CH₂—COOH benzyl C₃₁H₃₀FN₇O₃ 568.24 568.6 31 3-F ethoxy —CH₂—COOH benzylC₃₀H₂₈FN₇O₄ 570.22 570.6 32 — ethoxy —CH₂—COOH benzyl C₃₀H₂₉N₇O₄ 552.23552.4 33 — propyl —CH₂COOH 2-F- C₃₁H₃₀FN₇O₃ 568.24 568.2 benzyl 34 —propyl —CH₂COOH 2-Cl- C₃₁H₃₀ClN₇O₃ 584.21 584.2 benzyl 35 — propyl—CH₂COOH 2-CH₃- C₃₂H₃₃N₇O₃ 564.26 564.6 benzyl 36 — propyl —CH₂COOH2-CF₃- C₃₂H₃₀F₃N₇O₃ 618.24 618.6 benzyl 37 3-F ethyl —CH(OH)—COOH benzylC₃₀H₂₈FN₇O₄ 570.22 570.2 38 3-F ethoxy —CH₂SH i-butyl C₂₆H₃₀FN₇O₂S524.22 524.4 1.5-Ethoxy-1-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 2.1-[3-Fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 3.1-[3-Fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-mercaptomethyl-3-methylbutyl)amide 4.5-Ethoxy-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 5.5-Ethoxy-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-mercaptomethyl-3-methyl- butyl)amide 6.5-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 7.5-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-mercaptomethyl-3-methyl- butyl)amide 8.(2R,3R)-3-({5-Ethoxy-1-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid 9.(2R,3R)-3-({5-Ethoxy-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid 10.(2R,3R)-3-(1-((2-Fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 11.1-((2-Fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-N-((R)-1-mercapto-3-phenylpropan-2-yl)-5-propyl-1H-pyrazole-3-carboxamide12.(2R,3R)-2-Hydroxy-4-phenyl-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid methyl ester 13.(R)-4-(2-Bromophenyl)-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid 14.5-Propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid [(R)-1-benzyl-2-(formylhydroxyamino)ethyl]amide 15.5-Propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid [(R)-2-(2-chlorophenyl)-1-hydroxycarbamoylmethylethyl]amide 16.5-Propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-hydroxycarbamoylmethyl-2-phenylethyl)amide 17.1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-N-((2R,3R)-3-hydroxy-4-(hydroxyamino)-4-oxo-1-phenylbutan-2-yl)-5-propyl-1H-pyrazole-3-carboxamide 18.(R)-4-(2-Chlorophenyl)-3-({1-[2-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)butyricacid 19.(R)-3-({1-[2-Fluoro-2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-(2-trifluoromethylphenyl)butyricacid 20.(R)-4-(2-chlorophenyl)-3-(1-((3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)butanoicacid 21.(R)-3-(1-((3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-4-(2-(trifluoromethyl)phenyl)butanoicacid 22.(2R,3R)-3-(1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-4-(2-chlorophenyl)-2-hydroxybutanoicacid 23.(2R,3R)-3-(1-((3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 24.(2R,3R)-3-(1-((2-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 25.5-Propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid {(R)-1-[(formylhydroxyamino)methyl]-3-methylbutyl}amide 26.(2R,3R)-3-(1-((3,5-difluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 27.(R)-4-Phenyl-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid methyl ester 28.(R)-4-(2-Chlorophenyl)-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid methyl ester 29.(R)-3-({5-Propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-4-o-tolyl-butyricacid methyl ester 30.(R)-3-({1-[3-Fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 31.(R)-3-({5-Ethoxy-1-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 32.(R)-3-({5-Ethoxy-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 33.(R)-4-(2-Fluorophenyl)-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid 34.(R)-4-(2-Chlorophenyl)-3-({5-propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)butyricacid 35.(R)-3-({5-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-4-o-tolylbutyricacid 36.(R)-3-({5-Propyl-1-[2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-4-(2-trifluoromethylphenyl)butyricacid 37.(2R,3R)-3-({5-Ethyl-1-[3-fluoro-2′-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid 38.5-Ethoxy-1-[3-fluoro-2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid ((R)-1-mercaptomethyl-3-methylbutyl)amide

Preparation 52-Bromo-N-[1-dimethylaminometh-(E)-ylidene]benzenesulfonamide

1,1-Dimethoxy-N,N-dimethylmethanamine (14.6 mL, 104 mmol) was added to asolution of 2-bromobenzene-1-sulfonamide (20.4 g, 86.4 mmol) in DMF (56mL, 720 mmol) and the resulting solution was stirred at room temperaturefor 90 minutes. A solution of sodium hydrogen sulfate (1.7 g, 14 mmol)in water (170 mL, 9.4 mol) was cooled at 0° C. and then added to thereaction mixture. The precipitate was filtered, washed with water, anddried to yield the title compound (24.3 g) as a white solid.

Preparation 6 4′-Methylbiphenyl-2-sulfonic acid1-dimethylaminometh-(E)-ylideneamide

2-Bromo-N-[1-dimethylaminometh-(E)-ylidene]-benzenesulfonamide (5.4 g,18.4 mmol), 4-methylphenylboronic acid (5.0 g, 36.8 mmol) and potassiumcarbonate (5.1 g, 36.8 mmol) were dissolved in water (19.7 mL, 1090mmol), EtOH (49.2 mL, 842 mmol) and toluene (98.3 mL, 923 mmol). Theresulting mixture was stirred under nitrogen.Tetrakis(triphenylphosphine)palladium(0) (1.4 g, 1.2 mmol) was added.The mixture was heated at 60° C. for 115 minutes, at 70° C. for 30minutes, then cooled to room temperature. Water (100 mL) and EtOAc (100mL) were added. The mixture was washed with saturated aqueous NaCl,extracted with EtOAc, dried over MgSO₄, filtered, and concentrated toyield a red solid. The product was triturated with 1:1 EtOAc:hexanes,filtered, and rinsed with hexanes to yield a reddish-brown solid. Theproduct was triturated with EtOAc, filtered, and rinsing with EtOAc toyield the title compound (4.6 g) as a light brown solid.

Preparation 7 4′-bromomethylbiphenyl-2-sulfonic acid1-dimethylaminometh-(E)-ylideneamide

4′-Methylbiphenyl-2-sulfonic acid 1-dimethylaminometh-(E)-ylideneamide(540.0 mg, 1786 μmol), NBS (318 mg, 1.8 mmol), and benzoyl peroxide (4.3mg, 17.8 μmol) were dissolved in chlorobenzene (7.0 mL, 69 mmol) and theresulting solution was heated at 100° C. for 90 minutes. The mixture wascooled to room temperature and water was added. The mixture wasextracted with DCM, washed with saturated NaHCO₃ and saturated aqueousNaCl, extracted again with DCM, dried over MgSO4, filtered, andconcentrated. The crude product was purified by flash chromatography (40g, 0-100% EtOAc in hexanes), then taken up in EtOAc (4.5 mL) and DCM(1.5 mL). Additional DCM (3.0 mL) was added and the mixture was heatedat 60° C. The mixture was cooled in the freezer overnight, thenconcentrated. The material was taken up in DCM (2 mL), EtOAc (6 mL) wasadded, and the resulting solution placed in the freezer. A precipitateformed and was filtered to yield the title compound (279 mg) as a whitesolid.

Preparation 81-(2′-Acetylsulfamoyl-biphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid

Ethyl 3-n-propylpyrazole-5-carboxylate (1.3 g, 7.2 mmol), 4′-bromomethylbiphenyl-2-sulfonic acid 1-dimethylaminometh-(E)-ylideneamide (6.2 g,7.2 mmol) and potassium carbonate (2.0 g, 14.3 mmol) were dissolved inDMF (50 mL, 700 mmol) and the resulting mixture was stirred at roomtemperature until the reaction was nearly complete (1:1 mixture ofregioisomers). The mixture was partially concentrated, then partitionedbetween water and EtOAc. The EtOAc layer was then washed with saturatedaqueous NaCl, dried over Na₂SO₄ and concentrated. The crude product waspurified by flash chromatography (0-50% EtOAc/hexanes, dry load) toyield1-(2′-{[1-dimethyl-aminometh-(E)-ylidene]sulfamoyl}biphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ethyl ester (1.2 g).

To a solution of1-(2′-{[1-dimethylaminometh-(E)-ylidene]sulfamoyl}biphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ethyl ester (1.2 g, 2.5 mmol) in isopropyl alcohol (15.0 mL, 196mmol) was added 12 M of HCl in water (3.9 mL, 46.6 mmol), and theresulting solution was heated to reflux for 5 hours. The mixture wascooled to room temperature and stirred overnight. The solution wasadjusted to a pH of 5 using 1 N NaOH. The resulting suspension wasfiltered. The filtrate was concentrated to remove the isopropyl alcoholand the aqueous layer was extracted with EtOAc and DCM. The combinedextracts were dried over MgSO₄, filtered and concentrated.

The residue (890 mg) was dissolved in methylene chloride (13.1 mL, 204mmol). DIPEA (5.2 mL, 29.8 mmol) and acetic anhydride (2.4 mL, 24.9mmol) was added and the resulting solution was stirred overnight. Themixture was concentrated and the residue was diluted with t-butylalcohol (30 mL, 300 mmol). 0.20 M LiOH in water (60 mL, 10 mmol) wasadded and the mixture was stirred overnight. The mixture wasconcentrated and then extracted with EtOAc. The EtOAc was back-extractedwith 1 N LiOH. The combined aqueous layers were acidified with 1 N HClto pH 4-5. The acidic aqueous layer was extracted with EtOAc and DCM toyield the title compound (1.0 g).

Example 4(2R,3R)-3-(1-((2′-(N-Acetylsulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicAcid

A solution of1-(2′-acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid (250 mg, 570 μmol), HATU (215 mg, 566 μmol) and DIPEA (592 μL, 3.4mmol) in DMF (18 mL, 230 mmol) was stirred to pre-activate the acid.After 15 minutes, (2R,3R)-3-amino-2-hydroxy-4-phenylbutyric acid (110mg, 566 μmol) was added The resulting solution was stirred at 45° C.overnight. The mixture was concentrated, re-dissolved in water/MeCN/TFAand purified using reverse phase liquid chromatography to yield thetitle compound (197 mg, 97% purity). MS m/z: [M+H]⁺ calcd forC₃₂H₃₄N₄O₇S, 619.22; found 619.6.

Preparation 9N-t-Butyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide

2-Bromobenzenesulfonyl chloride (100.9 g, 394.9 mmol) was dissolved inmethylene chloride (500 mL, 8.0 mol) and cooled at 0° C. t-Butylamine(41.3 mL, 395 mmol) was added in 3 portions over approximately 1 minute.DIPEA (75.7 mL, 434 mmol) was immediately added in 3 portions overapproximately 1 minute. The mixture was warmed to room temperature andstirred overnight. The product was washed with 1M H₃PO₄ (2×), withsaturated. NaHCO₃, and with saturated aqueous NaCl, then dried overMgSO4, filtered, and concentrated to yield2-bromo-N-t-butyl-benzenesulfonamide (112 g) as a light brown solid.

2-Bromo-N-t-butyl-benzenesulfonamide (10.0 g, 34.2 mmol) was mixed withpalladium acetate (0.768 g, 3.42 mmol). Potassium acetate (13.4 g, 137mmol) was added followed by bis(pinacolato)diboron (10.4 g, 41.1 mmol)then DMF (265 mL, 3420 mmol). The resulting mixture was stirred undernitrogen, heated to reflux for 2 hours, then heated at 70° C. for 48hours. The mixture was poured onto ice, partitioned with EtOAc (200 mL),the organics were washed with saturated aqueous NaCl, dried over MgSO₄,filtered and concentrated. The product was purified by flashchromatography in hexanes:EtOAc 0-75% to yield the title compound (6.3g).

Preparation 105-Propyl-1-(2′-sulfamoylbiphenyl-4-ylmethyl)-1H-pyrazole-3-carboxylicAcid Ethyl Ester

1-(4-Bromobenzyl)-5-propyl-1H-pyrazole-3-carboxylic acid ethyl ester(1.2 g, 3.3 mmol) andN-t-butyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (1.4 g, 4.0 mmol) were combined with toluene (70.9 mL, 666.0mmol) and EtOH (18.5 mL, 316.7 mmol). Potassium carbonate (920 mg, 6.7mmol) was dissolved in water (9.6 mL, 532.8 mmol) and added to themixture, and stirred. Tetrakis(triphenyl-phosphine)palladium(0) (385 mg,333 μmol) was added quickly and the mixture heated at 100° C. for 30minutes in the microwave. EtOAc and water was added and organics wereextracted and evaporated. The crude product was dissolved in neat TFA(20 mL, 300 mmol) and stirred at 50° C. for 1.5 hours. The product wasdried under vacuum. DCM (20 mL) and saturated NaHCO₃ (20 mL) were added,stirred and extracted, dried, and evaporated to yield the titlecompound.

Preparation 111-((2′-(N-(methoxycarbonyl)sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxylicacid

5-Propyl-1-(2′-sulfamoylbiphenyl-4-ylmethyl)-1H-pyrazole-3-carboxylicacid ethyl ester (80 mg, 0.2 mmol) was dissolved in methylene chloride(2.0 mL, 30.6 mmol). Methyl chloroformate (17.4 μL, 224 μmol) was added,along with DIPEA (81.5 μL, 468 μmol). The mixture was stirred at roomtemperature for 2 hours. 1 M LiOH in water (1.5 mL, 1.50 mmol) andTHF:EtOH 2:1 mL were added and the resulting mixture was stirred for 2hours. The reaction was quenched with 1 N HCl and DCM (4 mL) was added.The organics were extracted and dried under vacuum to yield the titlecompound.

Example 5(2R,3R)-2-hydroxy-3-(1-((2′-(N-(methoxycarbonyl)-sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-4-phenylbutanoicAcid

A solution of1-((2′-(N-(methoxycarbonyl)sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxylicacid (40.2 mg, 87.8 μmol), DIPEA (91.8 μL, 527 μmol) and HATU (33.4 mg,87.8 μmol) in DMF (0.4 mL, 4 mmol) was stirred to pre-activate the acid.After 1 minute, (2R,3R)-3-amino-2-hydroxy-4-phenylbutyric acid (17.1 mg,87.8 μmol) was added, and the mixture was stirred for 10 minutes. Thereaction was quenched by adding 1 N HCl and extracted with DCM. Theorganic layer was added to AcOH (1 mL) and evaporated in vacuo. Thematerial was then purified by preparative HPLC to yield the titlecompound (10 mg, 95% purity). MS m/z: [M+H]⁺ calcd for C₃₂H₃₄N₄O₈S,635.21; found 635.20.

Example 6

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds6-1 to 6-28, having the following formula, were prepared:

MS m/z: [M + H]⁺ # R R³ R⁴ R⁵ Formula calcd found 1 3-F ethoxy —CH₂SHbenzyl C₃₀H₃₁FN₄O₅S₂ 611.17 611.4 2 — ethoxy —CH₂SH benzyl C₃₀H₃₂N₄O₅S₂593.18 593.6 3 — ethoxy —CH(OH)—COOH benzyl C₃₁H₃₂N₄O₈S 621.19 621.6 43-F ethoxy —CH(OH)—COOH benzyl C₃₁H₃₁FN₄O₈S 639.18 639.4 5 — propyl—CH₂SH benzyl C₃₁H₃₄N₄O₄S₂ 591.20 591.6 6 2-F propyl —CH₂SH benzylC₃₁H₃₃FN₄O₄S₂ 609.19 609.4 7 2-F propyl —CH(OH)—COOH benzyl C₃₂H₃₃FN₄O₇S637.21 637.6 8 — propyl —CH₂COOH 2-Br- C₃₂H₃₃N₄O₆S 681.13 681.4 benzyl 9— propyl —CH(OH)—COOH 2-Cl- C₃₂H₃₃ClN₄O₇S 653.18 653.6 benzyl 10 3-Fethyl —CH(OH)—COOH benzyl C₃₁H₃₁FN₄O₇S 623.19 623.2 11 3-F butyl—CH(OH)—COOH benzyl C₃₃H₃₅FN₄O₇S 651.22 651.2 12 — propyl —CH(OH)—COOHbenzyl C₃₄H₃₈N₄O₇S 647.25 647.2 13 — propyl —CH₂N(OH)—C(O)H benzylC₃₂H₃₅N₅O₆S 618.23 618.4 14 3-F propyl —CH(OH)—COOH benzyl C₃₂H₃₃FN₄O₇S637.21 637.4 15 — propyl —CH₂N(OH)—C(O)H i-butyl C₂₉H₃₇N₅O₆S 584.25584.4 16 3-F propyl —CH₂C(O)—NH(OH) benzyl C₃₂H₃₄FN₅O₆S 636.22 636.2 172-F propyl —CH₂C(O)—NH(OH) benzyl C₃₂H₃₄FN₅O₆S 636.22 636.2 18 — propyl—CH₂C(O)—NH(OH) benzyl C₃₂H₃₅N₅O₆S 618.23 618.6 19 3,5- propyl—CH(OH)—COOH benzyl C₃₂H₃₂F₂N₄O₇S 655.20 655.2 diF 20 3,5- propyl—CH₂C(O)—NH(OH) benzyl C₃₂H₃₃F₂N₅O₆S 654.21 654.6 diF 21 2-F propyl—CH₂C(O)—NH(OH) i-butyl C₂₉H₃₆FN₅O₆S 602.24 602.6 22 — propyl—CH₂N(OH)—C(O)H benzyl C₃₂H₃₅N₅O₆S 618.23 618.6 23 — propyl—CH₂N(OH)—C(O)H i-butyl C₂₉H₃₇N₅O₆S 584.25 584.6 24 3-F ethoxy —CH₂COOHbenzyl C₃₁H₃₁FN₄O₇S 623.19 623.6 25 — propyl —CH₂COOH 2-CF₃-C₃₃H₃₃F₃N₄O₆S 671.21 671.4 benzyl 26 — propyl —CH₂COOH 2-CH₃-C₃₃H₃₆N₄O₆S 617.24 617.6 benzyl 27 — propyl —CH₂COOH 2-Cl- C₃₂H₃₃ClN₄O₆S637.18 637.4 benzyl 28 — propyl —CH₂COOH benzyl C₃₂H₃₄N₄O₆S 603.22603.6 1.1-(2′-Acetylsulfamoyl-3-fluorobiphenyl-4-ylmethyl)-5-ethoxy-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 2.1-(2′-Acetylsulfamoyl-biphenyl-4-ylmethyl)-5-ethoxy-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercapto- ethyl)amide 3.(2R,3R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-ethoxy-1H-pyrazole-3-carbonyl]amino}-2-hydroxy-4-phenyl-butyricacid 4.(2R,3R)-3-{[1-(2′-Acetylsulfamoyl-3-fluorobiphenyl-4-ylmethyl)-5-ethoxy-1H-pyrazole-3-carbonyl]amino}-2-hydroxy-4-phenylbutyricacid 5.1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercapto- ethyl)amide 6.1-(2′-Acetylsulfamoyl-2-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 7.(2R,3R)-3-{[1-(2′-Acetylsulfamoyl-2-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-2-hydroxy-4-phenylbutyricacid 8.(R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-4-(2-bromophenyl)butyricacid 9.(2R,3R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]-amino}-4-(2-chlorophenyl)-2-hydroxy-butyricacid 10.(2R,3R)-3-{[1-(2′-Acetylsulfamoyl-3-fluoro-biphenyl-4-ylmethyl)-5-ethyl-1H-pyrazole-3-carbonyl]amino}-2-hydroxy-4-phenylbutyricacid 11.(2R,3R)-3-{[1-(2′-Acetylsulfamoyl-3-fluorobiphenyl-4-ylmethyl)-5-butyl-1H-pyrazole-3-carbonyl]amino}-2-hydroxy-4-phenylbutyricacid 12.(2R,3R)-2-Hydroxy-3-{[1-(2′-isobutyrylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-4-phenylbutyric acid 13.1-(2′-Acetylsulfamoyl-biphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid [(R)-1-benzyl-2-(formyl-hydroxy-amino)-ethyl]amide 14.(2R,3R)-3-(1-((2′-(N-acetylsulfamoyl)-3-fluorobiphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 15.1-(2′-Acetylsulfamoyl-biphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid {(R)-1-[(formyl-hydroxy-amino)-methyl]-3-methylbutyl}amide 16.1-(2′-Acetylsulfamoyl-3-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-hydroxycarbamoylmethyl-2-phenylethyl)amide 17.1-(2′-Acetylsulfamoyl-2-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-hydroxycarbamoylmethyl-2-phenylethyl)amide 18.1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-hydroxycarbamoylethyl)amide 19.(2R,3R)-3-(1-((2′-(N-acetylsulfamoyl)-3,5-difluorobiphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 20.1-(2′-Acetylsulfamoyl-3,5-difluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-hydroxycarbamoylmethyl-2-phenylethyl)amide 21.1-(2′-Acetylsulfamoyl-2-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylic acid (1-hydroxycarba-moylmethyl-3-methylbutyl)amide 23.1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid [(S)-1-benzyl-2-(formylhydroxyamino)ethyl]amide 23.1-(2′-Acetylsulfamoyl-biphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid {(S)-1-[(formylhydroxyamino)methyl]-3-methylbutyl}amide 24.(R)-3-{[1-(2′-Acetylsulfamoyl-3-fluorobiphenyl-4-ylmethyl)-5-ethoxy-1H-pyrazole-3-carbonyl]amino}-4-phenylbutyricacid 25.(R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-4-(2-trifluoromethylphenyl)butyricacid 26.(R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-4-o-tolylbutyricacid 27.(R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-4-(2-chlorophen-yl)butyric acid 28.(R)-3-{[1-(2′-Acetylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-4-phenylbutyricacid

Example 7

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds7-1 to 7-22, having the following formula, were prepared:

MS m/z: [M + H]⁺ # R^(1a) R⁴ R⁵ Formula calcd found 1 —NH(CH₂CH₃)—CH(OH)—COOH benzyl C₃₃H₃₇N₅O₇S 648.24 648.2 2 cyclopropyl —CH(OH)—COOHbenzyl C₃₄H₃₆N₄O₇S 645.23 645.2 3 2F-phenyl —CH(OH)—COOH benzylC₃₇H₃₅FN₄O₇S 699.22 699.2 4 4-pyridyl —CH(OH)—COOH benzyl C₃₆H₃₅N₅O₇S682.23 682.2 5 -3-isoxazolyl-5- —CH(OH)—COOH benzyl C₃₅H₃₅N₅O₈S 686.22686.2 methyl 6 -5-isoxazolyl —CH(OH)—COOH benzyl C₃₄H₃₃N₅O₈S 672.21672.2 7 —OCH₂CH₃ —CH(OH)—COOH benzyl C₃₃H₃₆N₄O₈S 649.23 649.2 8 —CH₂OCH₃—CH(OH)—COOH benzyl C₃₃H₃₆N₄O₈S 649.23 649.2 9 2-pyridyl —CH(OH)—COOHbenzyl C₃₆H₃₅N₅O₇S 682.23 682.2 10 ethyl —CH(OH)—COOH benzyl C₃₃H₃₆N₄O₇S633.23 635.2 11 —NH(CH₃) —CH(OH)—COOH benzyl C₃₂H₃₅N₅O₇S 634.23 634.2 12phenyl —CH(OH)—COOH benzyl C₃₇H₃₆N₄O₇S 681.23 681.2 13 —CH(CH₃)OH—CH(OH)—COOH benzyl C₃₃H₃₆N₄O₈S 649.23 649.2 14 —C(CH₃)₂OH —CH(OH)—COOHbenzyl C₃₄H₃₈N₄O₈S 663.24 663.2 15 —CH₂OH —CH(OH)—COOH benzylC₃₂H₃₄N₄O₈S 635.21 635.2 16 —CH(CH₃)OH —CH(OH)—COOH benzyl C₃₃H₃₆N₄O₈S649.23 649.2 17 —(CH₂)₂—OCH₃ —CH(OH)—COOH benzyl C₃₄H₃₈N₄O₈S 663.24663.2 18 —CH₂—OCH₃ —CH₂COOH 2-Cl- C₃₃H₃₅ClN₄O₇S 667.19 667.2 benzyl 19—C(CH₃)₂NH₂ —CH(OH)—COOH benzyl C₃₄H₃₉N₅O₇S 662.26 662.4 20 —N(CH₃)₂—CH(OH)—COOH benzyl C₃₃H₃₇N₅O₇S 648.24 648.6 21 1-pyrrolidyl—CH(OH)—COOH benzyl C₃₅H₃₉N₅O₇S 674.26 674.6 22 4-morpholinyl—CH(OH)—COOH benzyl C₃₅H₃₉N₅O₈S 690.25 690.4 1.(2R,3R)-3-(1-((2′-(N-(ethylcarbamoyl)sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 2.(2R,3R)-3-({1-[2′-(Cyclopropanecarbonylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid 3.(2R,3R)-3-({1-[2′-(2-Fluorobenzoylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid 4.(2R,3R)-2-Hydroxy-4-phenyl-3-[(5-propyl-1-{2′-[(pyridine-4-carbonyl)sulfamoyl]biphenyl-4-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid 5.(2R,3R)-2-Hydroxy-3-[(1-{2′-[(5-methylisoxazole-3-carbonyl)sulfamoyl]biphenyl-4-ylmethyl}-5-propyl-1H-pyrazole-3-carbonyl)amino]-4-phenylbutyricacid 6.(2R,3R)-2-Hydroxy-3-[(1-{2′-[(isoxazole-5-carbonyl)sulfamoyl]biphenyl-4-ylmethyl}-5-propyl-1H-pyrazole-3-carbonyl)amino]-4-phenylbutyricacid 7.(2R,3R)-3-(1-((2′-(N-(ethoxycarbonyl)sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 8.(2R,3R)-2-Hydroxy-3-({1-[2′-(2-methoxyacetylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 9.(2R,3R)-2-Hydroxy-4-phenyl-3-[(5-propyl-1-{2′-[(pyridine-2-carbonyl)sulfamoyl]biphenyl-4-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid 10.(2R,3R)-2-Hydroxy-4-phenyl-3-{[1-(2′-propionylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}butyricacid 11.(2R,3R)-2-hydroxy-3-(1-((2′-(N-(methylcarbamoyl)sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-4-phenylbutanoicacid 12.(2R,3R)-3-{[1-(2′-Benzoylsulfamoylbiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carbonyl]amino}-2-hydroxy-4-phenylbutyricacid 13.(2R,3R)-2-Hydroxy-3-({1-[2′-((S)-2-hydroxypropionylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 14.(2R,3R)-2-Hydroxy-3-({1-[2′-(2-hydroxy-2-methylpropionylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 15.(2R,3R)-2-Hydroxy-3-({1-[2′-(2-hydroxyacetylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 16.(2R,3R)-2-Hydroxy-3-({1-[2′-((R)-2-hydroxypropionylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 17.(2R,3R)-2-Hydroxy-3-({1-[2′-(3-methoxy-propionylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 18.(R)-4-(2-Chlorophenyl)-3-({1-[2′-(2-methoxyacetylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)butyricacid 19.(2R,3R)-3-({1-[2′-(2-Amino-2-methylpropionylsulfamoyl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid 20.(2R,3R)-3-(1-((2′-(N-(dimethylcarbamoyl)sulfamoyl)biphenyl-4-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid 21.(2R,3R)-2-Hydroxy-4-phenyl-3-[(5-propyl-1-{2′-[(pyrrolidine-1-carbonyl)sulfamoyl]biphenyl-4-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid 22.(2R,3R)-2-Hydroxy-3-[(1-{2′-[(morpholine-4-carbonyl)sulfamoyl]biphenyl-4-ylmethyl}-5-propyl-1H-pyrazole-3-carbonyl)amino]-4-phenylbutyricacid

Preparation 121-(2′-t-Butoxycarbonyl-3-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicAcid

Ethyl 3-n-propylpyrazole-5-carboxylate (1.05 g, 5.8 mmol),4′-bromomethyl-3′-fluorobiphenyl-2-carboxylic acid t-butyl ester (2.0 g,5.8 mmol), and potassium carbonate (1.6 g, 11.5 mmol) were dissolved inDMF (10 mL). The mixture was stirred at 65° C. for 16 hours, thendiluted with EtOAc. The organic layer was washed with saturated NaHCO₃and saturated aqueous NaCl, and dried over Na₂SO₄. The solvent wasremoved in vacuo and the residue was purified by flash chromatography(EtOAc in hexanes) to yield1-(2′-t-butoxycarbonyl-3-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ethyl ester (2.7 g).

1-(2′-t-Butoxycarbonyl-3-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ethyl ester (758 mg, 1.6 mmol) was diluted with MeOH (10 mL, 0.2mol), and LiOH (233 mg, 4.9 mmol) was added. The mixture was stirredovernight then concentrated to dryness. The residue was dissolved in DCMand water and the aqueous layer was neutralized with AcOH. The waterlayer was extracted with DCM and the combined organic layers were washedwith saturated aqueous NaCl and dried over Na₂SO₄. The crude product waspurified by flash chromatography (EtOAc in hexanes) to yield the titlecompound (710 mg).

Example 84′-[3-((R)-1-Benzyl-2-mercaptoethylcarbamoyl)-5-propylpyrazol-1-ylmethyl]-3′-fluorobiphenyl-2-carboxylic Acid

1-(2′-t-Butoxycarbonyl-3-fluorobiphenyl-4-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid (150 mg, 342 μmol) was dissolved into DMF (5 mL). HATU (130 mg, 342μmol) was added and the mixture was stirred for 15 minutes.(R)-2-((R)-2-Amino-3-phenylpropyldisulfanyl)-1-benzylethylamine.2[HCl](69.3 mg, 171 μmol) was added followed by DIPEA (149 μL, 855 μmol). Themixture was stirred at room temperature for 5 minutes then warmed to 45°C. After 5 hours, the mixture was cooled to room temperature andpartitioned between EtOAc (25 mL) and 10% LiCl (10 mL). The organic wasthen washed with saturated NaHCO₃ (10 mL), saturated aqueous NaCl, driedover MgSO₄ and was concentrated to yield a brown oil. The mixture waschromatographed with 1:1 EtOAc:hexanes to obtain the dimer intermediate,which was dissolved in 30% TFA:DCM (10 mL) and stirred for 3 hours atroom temperature. The mixture was concentrated under reduced pressureand dried under high vacuum. The material was redissolved in THF (5 mL),and a solution of tris(2-carboxyethyl)phosphine hydrochloride (120 mg,430 μmol) in water (0.5 mL) was added. The resulting mixture was stirredat room temperature for 3 hours, then concentrated and dissolved in 1:1AcOH/water. The crude solution was filtered and chromatographed (1:1water/MeCN with 0.1% TFA) to obtain the title compound (100 mg). MS m/z:[M+H]⁺ calcd for C₃₀H₃₀FN₃O₃S, 532.20; found 532.4.

Example 9

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds9-1 to 9-7, having the following formula, were prepared:

MS m/z: [M + H]⁺ # R R³ R⁴ R⁵ Formula calcd found 1 3-F propyl —CH₂SHi-butyl C₂₇H₃₂FN₃O₃S 498.22 498.6 2 3-F ethoxy —CH₂SH benzylC₂₉H₂₈FN₃O₄S 534.18 534.4 3 3-F butyl —CH₂SH i-butyl C₂₈H₃₄FN₃O₃S 512.23512.6 4 3-F butyl —CH₂SH benzyl C₃₁H₃₂FN₃O₃S 546.22 546.6 5 — propylCH(OH)—COOH benzyl C₃₁H₃₁N₃O₆ 542.22 542.8 6 — propyl —CH₂SH benzylC₃₀H₃₁N₃O₃S 514.21 514.6 7 2,3-diF propyl CH(OH)—COOH benzylC₃₁H₂₉F₂N₃O₆ 578.20 578.6 1.3′-Fluoro-4′-[3-((R)-1-mercaptomethyl-3-methylbutylcarbamoyl)-5-propylpyrazol-1-ylmethyl]biphenyl-2-carboxylicacid 2.4′-[3-((R)-1-Benzyl-2-mercaptoethylcarbamoyl)-5-ethoxypyrazol-1-ylmethyl]-3′-fluorobiphenyl-2-carboxylicacid 3.4′-[5-Butyl-3-((R)-1-mercaptomethyl-3-methylbutylcarbamoyl)-pyrazol-1-ylmethyl]-3′-fluorobiphenyl-2-carboxylicacid 4.4′-[3-((R)-1-Benzyl-2-mercaptoethylcarbamoyl)-5-butylpyrazol-1-ylmethyl]-3′-fluorobiphenyl-2-carboxylicacid 5.4′-[3-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxyethylcarbamoyl)-5-propylpyrazol-1-ylmethyl]biphenyl-2-carboxylicacid 6.4′-[3-((R)-1-Mercaptomethyl-2-phenylethylcarbamoyl)-5-propylpyrazol-1-ylmethyl]biphenyl-2-carboxylicacid 7.4′-[3-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxyethylcarbamoyl)-5-propylpyrazol-1-ylmethyl]-2′,3′-di-fluorobiphenyl-2-carboxylic acid

Preparation 135-Propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carboxylicAcid

A solution of (tetrazol-5-yl)phenylboronic acid (180 mg, 0.97 mmol),1-(6-bromopyridin-3-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylic acidethyl ester (285 mg, 809 μmol), tetrakis(triphenylphosphine)palladium(0)(50 mg, 40 μmol), 1.0 M NaOH in water (3.2 mL, 3.2 mmol) and MeOH (4.0mL, 100 mmol) was sparged shortly, capped under nitrogen and heated inthe microwave at 90° C. for 2 hours. The mixture was filtered to removethe Pd (rinsed with MeOH). The filtrate was concentrated to remove theMeOH, then extracted with EtOAc. The organic layer was extracted with 1N NaOH. The combined aqueous layers were acidified with 1 N HCl to pH3-4 and extracted with EtOAc. The organics were dried over MgSO₄,filtered and concentrated to yield the title compound (122 mg), whichwas used without further purification.

Example 10(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricAcid

A solution of5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carboxylicacid (20 mg, 0.05 mmol), HATU (19.5 mg, 0.0514 mmol) and DIPEA (44.7 uL,257 μmol) in DMF (1.59 mL, 20.5 mmol) was stirred to pre-activate theacid. After 30 minutes, (R)-3-amino-4-(2-chlorophenyl)butanoic acid.HCl(12.8 mg, 0.0514 mmol)) was added. The resulting solution was stirredovernight. The mixture was concentrated, re-dissolved in water/MeCN/TFAand purified using reverse phase liquid chromatography to yield thetitle compound (20.5 mg; 100% purity) as a TFA salt. MS m/z: [M+H]⁺calcd for C₃₀H₂₉ClN₈O₃, 585.21; found 585.4.

Example 11(R)-3-[(5-Propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]-pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)-amino]-4-(2-trifluoromethylphenyl)butyricAcid

A solution of5-propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carboxylicacid (20 mg, 0.05 mmol), HATU (19.5 mg, 0.0514 mmol) and DIPEA (44.7 uL,0.257 mmol) in DMF (1.59 mL, 20.5 mmol) was stirred to pre-activate theacid. After 30 minutes, (R)-3-amino-4-(2-trifluoromethylphenyl)butanoicacid.HCl (14.6 mg, 51.4 μmol)) was added. The resulting solution wasstirred overnight. The mixture was concentrated, re-dissolved inwater/MeCN/TFA and purified using reverse phase liquid chromatography toyield the title compound (21.1 mg; 100% purity) as a TFA salt. MS m/z:[M+H]⁺ calcd for C₃₁H₂₉F₃N₈O₃, 619.23; found 619.6.

Example 12(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid

A solution of5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carboxylicacid (40 mg, 103 μmol), HATU (39.0 mg, 0.103 mmol) and DIPEA (89.4 uL,514 μmol) in DMF (3.2 mL, 41.1 mmol) was stirred to pre-activate theacid. After 30 minutes, (R)-3-amino-4-(2-fluorophenyl)butanoic acid.HCl(24.0 mg, 103 μmol) was added. The resulting solution was stirredovernight. The mixture was concentrated, re-dissolved in water/MeCN/TFAand purified using reverse phase liquid chromatography to yield thetitle compound (15.7 mg; 100% purity) as a TFA salt. MS m/z: [M+H]⁺calcd for C₃₀H₂₉FN₈O₃, 569.23; found 569.2.

Preparation 141-(6-Bromopyridin-3-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylic AcidEthyl Ester

To a solution of 2-Bromo-5-methylpyridine (3.1 g, 17.4 mmol) in carbontetrachloride (40 mL, 400 mmol) was added benzoyl peroxide (230 mg, 950μmol) and NBS (3.4 g, 19.2 mmol). The resulting mixture was heated atreflux overnight. The mixture was cooled at 0° C. and the NBS wasremoved by filtration. The filtrate was concentrated to yield2-bromo-5-bromomethylpyridine (4.6 g), which was used directly in thenext step.

Ethyl 3-n-propylpyrazole-5-carboxylate (1.7 g, 9.1 mmol),2-bromo-5-bromomethylpyridine (4.6 g, 9.1 mmol) and potassium carbonate(1.8 g, 12.7 mmol) were dissolved in DMF (70.0 mL, 904 mmol). Theresulting suspension was stirred at room temperature overnight. Thematerial was diluted with aqueous. NaHCO₃ and diethyl ether. The aqueouslayer was further extracted with diethyl ether. The combined organicswere dried over MgSO₄, filtered and concentrated. The residue waspurified using flash chromatography [1^(st) 30:1 hexanes/EtOAc; 2^(nd)10:1 hexanes/EtOAc]. The two isomers were separated and analyzed by NMRand NOE: 1-(6-bromopyridin-3-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylicacid ethyl ester (1.2 g) and2-(6-bromopyridin-3-ylmethyl)-5-propyl-2H-pyrazole-3-carboxylic acidethyl ester.

Preparation 151-[6-(2-Acetylsulfamoylphenyl)pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicAcid

To a suspension ofN-t-butyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzenesulfonamide(722 mg, 2.1 mmol) and1-(6-bromopyridin-3-ylmethyl)-5-propyl-1H-pyrazole-3-carboxylic acidethyl ester (500 mg, 1.4 mmol) in toluene (4.0 mL, 37 mmol) was added asolution of potassium carbonate (392 mg, 2.84 mmol) in water (530 μL, 30mmol). The resulting solution was sparged shortly with nitrogen beforetetrakis(triphenylphosphine)-palladium(0) (328 mg, 284 μmol) was added.The mixture heated in the microwave at 100° C. for 30 minutes, thencooled to room temperature. Aqueous 1% citric acid was added, themixture was extracted with EtOAc. The combined organics were partiallyconcentrated, filtered through Celite® (EtOAc rinse), and concentrated.The crude residue was diluted with t-butyl alcohol (21.6 mL, 226 mmol),and 0.20 M LiOH in water (42.6 mL, 8.5 mmol) was added. The mixture wasstirred overnight, then concentrated and extracted with EtOAc. The EtOAcwas back-extracted with 1 N LiOH. The combined aqueous layers wereacidified with 1 N HCl to pH 4-5. The acidic aqueous layer was extractedwith EtOAc and DCM to yield1-[6-(2-t-butylsulfamoylphenyl)pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid (630 mg).

1-[6-(2-t-Butylsulfamoylphenyl)pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid (480 mg, 1.0 mmol) was dissolved in TFA (2.8 mL, 36.8 mmol) andheated at 40° C. for 3 hours. The mixture was diluted with toluene andconcentrated (azeotrope×3). The residue was dissolved in DCM (5.53 mL,86.2 mmol), then triethylamine (1.8 mL, 12.6 mmol) and acetic anhydride(992 μL, 10.5 mmol) were added. The resulting solution was stirred atroom temperature for 1 hour. Additional triethylamine and aceticanhydride were added to drive the reaction. The reaction was thenquenched with water (150 μL) and dried through azeotrope with toluene toyield the title compound (108 mg), which was used without furtherpurification.

Example 13(2R,3R)-3-({1-[6-(2-Acetylsulfamoylphenyl)pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricAcid

A solution of1-[6-(2-acetylsulfamoylphenyl)pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid (100 mg, 226 μmol) in DMF (7.0 mL, 90 mmol) was added to HATU (85.9mg, 226 μmol). The resulting mixture was stirred for 5 minutes before(2R,3R)-3-amino-2-hydroxy-4-phenylbutyric acid.HCl (52.4 mg, 226 μmol)and DIPEA (315 μL, 1.8 mmol) were added. The resulting solution washeated at 40° C. overnight. The mixture was concentrated, re-dissolvedin water/MeCN/TFA and purified using reverse phase liquid chromatographyto yield the title compound (48 mg; 91% purity) as a TFA salt. MS m/z:[M+H]⁺ calcd for C₃₁H₃₃N₅O₇S, 620.21; found 620.4.

Example 14

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds14-1 to 14-15, having the following formula were prepared:

MS m/z: [M + H]⁺ # R¹ R⁴ R⁵ Formula calcd found 1 1H-tetrazol-—CH(OH)—COOH benzyl C₃₀H₃₀N₈O₄ 567.24 567.6 5-yl 2 1H-tetrazol- —CH₂SHbenzyl C₂₉H₃₀N₈OS 539.23 539.6 5-yl 3 —SO₂NH—C(O)CH₃ —CH₂SH benzylC₃₀H₃₃N₅O₄S₂ 592.20 592.6 4 1H-tetrazol- —CH₂—COOH 2-Br-benzylC₃₀H₂₉BrN₈O₃ 629.15 631.4 5-yl 5 1H-tetrazol- —CH(OH)—COOH 2-Cl-benzylC₃₀H₂₉ClN₈O₄ 601.20 601.4 5-yl 6 1H-tetrazol- —CH₂—COOH benzylC₃₀H₃₀N₈O₃ 551.24 551.4 5-yl 7 1H-tetrazol- —CH₂—COOH i-butyl C₂₇H₃₂N₈O₃517.26 517.4 5-yl 8 1H-tetrazol- —CH(OH)—COOH i-butyl C₂₇H₃₂N₈O₄ 533.25533.2 5-yl 9 1H-tetrazol- —CH(OH)—COOH benzyl C₃₀H₃₀N₈O₄ 567.24 567.65-yl 10 1H-tetrazol- 5-yl —CH₂—COOH

C₂₈H₂₈N₈O₃S 557.20 557.2 11 1H-tetrazol- 5-yl —CH₂—COOH

C₂₈H₂₈N₈O₄ 541.22 541.2 12 1H-tetrazol- —CH₂—COOH 3-F-benzyl C₃₀H₂₉FN₈O₃569.23 569.2 5-yl 13 1H-tetrazol- —CH₂—COOH 4-F-benzyl C₃₀H₂₉FN₈O₃569.23 569.2 5-yl 14 —SO₂NH—C(O)CH₃ —CH₂—COOH 2-Cl-benzyl C₃₁H₃₂ClN₅O₆S638.18 638.4 15 —SO₂NH—C(O)CH₃ —CH₂—COOH benzyl C₃₁H₃₃N₅O₆S 604.22605.0 1.(2R,3R)-2-Hydroxy-4-phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 2.5-Propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide (TFA salt) 3.1-[6-(2-Acetylsulfamoyl-phenyl)pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide (TFA salt) 4.(R)-4-(2-Bromophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]-pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 5.(2R,3R)-4-(2-Chlorophenyl)-2-hydroxy-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyric acid (TFA salt) 6.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]-pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 7.(R)-5-Methyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)-amino]hexanoicacid (TFA salt) 8.(2R,3R)-2-Hydroxy-5-methyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]hexanoicacid d (TFA salt) 9.(2S,3R)-2-Hydroxy-4-phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 10.(R)-3-[(5-Propyl-1-{6-[2-(1H-tetrazol-5-yl)-phenyl]-pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)-amino]-4-thiophen-3-yl-butyricacid (TFA salt) 11.(R)-4-Furan-2-yl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 12.(R)-4-(3-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 13.(R)-4-(4-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 14.(R)-3-({1-[6-(2-Acetylsulfamoylphenyl)-pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}-amino)-4-(2-chlorophenyl)butyricacid (TFA salt) 15.(R)-3-({1-[6-(2-Acetylsulfamoylphenyl)-pyridin-3-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}-amino)-4-phenylbutyricacid (TFA salt)

Example 15

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds15-1 to 15-4, having the following formula were prepared:

MS m/z: [M + H]⁺ # R¹ R⁴ R⁵ Formula calcd found 1 1H-tetrazol-—CH(OH)—COOH benzyl C₃₀H₃₀N₈O₄ 567.24 567.6 5-yl 2 1H-tetrazol-—CH₂—COOH 2-Cl-benzyl C₃₀H₂₉ClN₈O₃ 585.21 585.4 5-yl 3 —SO₂NH—C(O)CH₃—CH(OH)—COOH benzyl C₃₁H₃₃N₅O₇S 620.21 620.6 4 —SO₂NH—C(O)CH₃ —CH₂—COOH2-Cl-benzyl C₃₁H₃₂ClN₅O₆S 638.18 638.4 1.(2R,3R)-2-Hydroxy-4-phenyl-3-[(5-propyl-1-{5-[2-(1H-tetrazol-5-yl)phenyl]pyridin-2-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 2.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{5-[2-(1H-tetrazol-5-yl)phenyl]pyridin-2-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 3.(2R,3R)-3-({1-[5-(2-Acetylsulfamoylphenyl)pyridin-2-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid (TFA salt) 4.(R)-3-({1-[5-(2-Acetylsulfamoylphenyl)pyridin-2-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-(2-chlorophenyl)butyricacid (TFA salt)

Example 16

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds16-1 to 16-7, having the following formula, were prepared:

MS m/z: [M + H]⁺ # R¹ R³ R⁴ R⁵ Formula calcd found 1

propyl —CH₂SH benzyl C₃₁H₃₁N₅O₃S 554.22 554.4 2

propyl —CH(OH)—COOH benzyl C₃₂H₃₁N₅O₆ 582.23 582.4 3

propyl —CH(OH)—COOH benzyl C₃₁H₃₁N₇O₅ 582.24 582.6 4

propyl —CH₂—COOH benzyl C₃₁H₃₁N₇O₄ 566.24 567.0 5

propyl —CH₂—COOH 2-Cl- benzyl C₃₁H₃₀ClN₇O₄ 600.21 600.6 6

propyl —CH(OH)—COOH 2-Cl- benzyl C₃₁H₃₀ClN₇O₅ 616.20 616.6 7

ethoxy —CH(OH)—COOH benzyl C₃₀H₂₉N₇O₆ 584.22 584.6 1.1-[2′-(5-Oxo-2,5-dihydro-[1,2,4]oxadiazol-3-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carboxylicacid ((R)-1-benzyl-2-mercaptoethyl)amide 2.(2R,3R)-2-Hydroxy-3-({1-[2′-(5-oxo-2,5-dihydro-[1,2,4]oxadiazol-3-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 3.(2R,3R)-2-Hydroxy-3-({1-[2′-(5-oxo-4,5-dihydrotetrazol-1-yl)-biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 4.(R)-3-({1-[2′-(5-Oxo-4,5-dihydrotetrazol-1-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid 5.(R)-4-(2-Chlorophenyl)-3-({1-[2′-(5-oxo-4,5-dihydrotetrazol-1-yl)-biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)butyricacid 6.(2R,3R)-4-(2-Chlorophenyl)-2-hydroxy-3-({1-[2′-(5-oxo-4,5-dihydrotetrazol-1-yl)biphenyl-4-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)butyric acid 7.(2R,3R)-3-({5-Ethoxy-1-[2′-(5-oxo-4,5-dihydrotetrazol-1-yl)-biphenyl-4-ylmethyl]-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid

Example 17

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds17-1 to 17-6, having the following formula were prepared:

MS m/z: [M + H]⁺ # R¹ R⁴ R⁵ Formula calcd found 1 1H-tetrazol- —CH₂—COOHbenzyl C₂₉H₂₉N₉O₃ 552.24 552.3 5-yl 2 1H-tetrazol- —CH₂—COOH 2-Cl-C₂₉H₂₈ClN₉O₃ 586.20 586.2 5-yl benzyl 3 1H-tetrazol- —CH(OH)—COOH benzylC₂₉H₂₉N₉O₄ 568.23 568.8 5-yl 4 —SO₂NH—C(O)CH₃ —CH₂—COOH benzylC₃₀H₃₂N6O₆S 605.21 605.8 5 —SO₂NH—C(O)CH₃ —CH(OH)—COOH benzylC₃₀H₃₂N₆O₇S 621 .2 1 621.8 6 —SO₂NH—C(O)CH₃ —CH₂—COOH 2-Cl-C₃₀H₃₁ClN6O₆S 639.17 639.4 benzyl 1.(R)-4-Phenyl-3-[(5-propyl-1-{5-[2-(1H-tetrazol-5-yl)phenyl]pyrazin-2-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 2.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{5-[2-(1H-tetrazol-5-yl)phenyl]pyrazin-2-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt) 3.(2R,3R)-3-(1-((5-(2-(1H-tetrazol-5-yl)phenyl)pyrazin-2-yl)methyl)-5-propyl-1H-pyrazole-3-carboxamido)-2-hydroxy-4-phenylbutanoicacid (TFA salt) 4.(R)-3-({1-[5-(2-Acetylsulfamoylphenyl)pyrazin-2-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-phenylbutyricacid (TFA salt) 5.(2R,3R)-3-({1-[5-(2-Acetylsulfamoylphenyl)pyrazin-2-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-2-hydroxy-4-phenylbutyricacid (TFA salt) 6.(R)-3-({1-[5-(2-Acetylsulfamoyl-phenyl)-pyrazin-2-ylmethyl]-5-propyl-1H-pyrazole-3-carbonyl}amino)-4-(2-chloro-phenyl)butyricacid (TFA salt)

Example 18

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compound18, having the following formula, was prepared:

MS m/z: [M + H]⁺ # R¹ R⁴ R⁵ Formula calcd found 1 tetra- —CH₂COOH 2-Cl-C₂₉H₂₈ClN₉O₃ 586.20 586.6 zole benzyl 1.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{2-[2-(1H-tetrazol-5-yl)phenyl]pyrimidin-5-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid (TFA salt)

Example 19

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds19-1 to 19-3, having the following formula were prepared:

These compounds are prodrugs of the compound of Example 3-16.

MS m/z: [M + H]⁺ # R^(4d) Formula calcd found 1 —C(O)CH₃ C₃₃H₃₄N₈O₄607.27 607.4 2 —C(O)-phenyl C₃₈H₃₆N₈O₄ 669.29 669.5 3—C(O)—CH(NH₂)[CH(CH₃)₂] C₃₆H₄₁N₉O₄ 664.33 664.4 1.5-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid [(R)-1-(acetoxycarbamoylmethyl)-2-phenylethyl]amide 2.5-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid [(R)-1-(benzoyloxycarbamoyl-methyl)-2-phenylethyl]amide 3.5-Propyl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-pyrazole-3-carboxylicacid{(R)-1-[((R)-2-amino-3-methyl-butyryloxycarbamoyl)methyl]-2-phenylethyl}amide

Example 20

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds20-1 to 20-11, having the following formula were prepared:

These compounds are prodrugs of the compound of Example 10.

MS m/z: [M + H]⁺ # R^(4c) Formula calcd found 1 —CH₂CH₃ C₃₂H₃₃ClN₈O₃613.24 613.2 2 —CH(CH₃)₂ C₃₃H₃₅ClN₈O₃ 627.25 627.7 3 —(CH₂)₃CH₃C₃₄H₃₇ClN₈O₃ 641.27 641.6 4 —(CH₂)₄CH₃ C₃₅H₃₉ClN₈O₃ 655.28 655.2 5—(CH₂)₂CH₃ C₃₃H₃₅ClN₈O₃ 627.25 627.4 6

C₃₆H₄₀ClN₉O₄ 698.29 698.6 7

C₃₅H₃₃ClN₈O₆ 697.22 697.2 8 —CH(CH₃)OC(O)O-cyclohexyl C₃₉H₄₃ClN₈O₆755.30 755.4 9 —CH(CH₃)OC(O)OCH₂CH₃ C₃₅H₃₇ClN₈O₆ 701.25 701.6 10—CH(CH₃)OC(O)OCH(CH₃)₂ C₃₆H₃₉ClN₈O₆ 715.27 715.6 11 —CH₃ C₃₁H₃₁ClN₈O₃599.22 599.4 1.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid ethyl ester (TFA salt)2.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid isopropyl ester (TFAsalt) 3.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid butyl ester (TFA salt)4.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid pentyl ester (TFAsalt) 5.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid propyl ester (TFAsalt) 6.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid 2-morpholin-4-yl-ethylester (TFA salt) 7.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid5-methyl-2-oxo-[1,3]dioxol-4-yl- methyl ester (TFA salt) 8.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid1-cyclohexyloxycarbonyloxy-ethyl ester (TFA salt) 9.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid1-ethoxycarbonyloxy-ethyl ester (TFA salt) 10.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid1-isopropoxycarbonyloxy-ethyl ester (TFA salt) 11.(R)-4-(2-Chlorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-yl-methyl}-1H-pyrazole-3-carbonyl)amino]butyric acid methyl ester (TFAsalt)

Example 21

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds21-1 to 21-6, having the following formula were prepared:

These compounds are prodrugs of the compound of Example 12.

MS m/z: [M + H]⁺ # R^(4c) Formula calcd found 1 —CH₂CH₃ C₃₂H₃₃FN₈O₃597.27 597.6 2 —(CH₂)₂CH₃ C₃₃H₃₅FN₈O₃ 611.28 611.4 3 —(CH₂)₃CH₃C₃₄H₃₇FN₈O₃ 625.30 625.6 4 —(CH₂)₄CH₃ C₃₅H₃₉FN₈O₃ 639.31 639.6 5—CH(CH₃)₂ C₃₃H₃₅FN₈O₃ 611.28 611.6 6 —CH₃ C₃₁H₃₁FN₈O₃ 583.25 583.4 1.(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid ethyl ester (TFA salt) 2.(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid propyl ester (TFA salt) 3.(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid butyl ester (TFA salt) 4.(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid pentyl ester (TFA salt) 5.(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid isopropyl ester (TFA salt) 6.(R)-4-(2-Fluorophenyl)-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid methyl ester (TFA salt)

Example 22

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compounds22-1 to 22-6, having the following formula were prepared:

These compounds are prodrugs of the compound of Example 14-6.

MS m/z: [M + H]⁺ # R^(4c) Formula calcd found 1 —CH(CH₃)₂ C₃₃H₃₆N₈O₃593.29 593.2 2 —(CH₂)₃CH₃ C₃₄H₃₈N₈O₃ 607.31 607.2 3 —(CH₂)₄CH₃C₃₅H₄₀N₈O₃ 621.32 621.2 4 —CH₂CH₃ C₃₂H₃₄N₈O₃ 579.28 579.6 5 —(CH₂)₂CH₃C₃₃H₃₆N₈O₃ 593.29 593.2 6 —CH₃ C₃₁H₃₂N₈O₃ 565.26 565.6 1.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid isopropyl ester (TFA salt) 2.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid butyl ester (TFA salt) 3.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid pentyl ester (TFA salt) 4.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid ethyl ester (TFA salt) 5.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid propyl ester (TFA salt) 6.(R)-4-Phenyl-3-[(5-propyl-1-{6-[2-(1H-tetrazol-5-yl)phenyl]pyridin-3-ylmethyl}-1H-pyrazole-3-carbonyl)amino]butyricacid methyl ester (TFA salt)

Example 23

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compound23-1 to 23-4 having the following formula can also be prepared:

# R¹ R⁴ R⁵ 1 tetrazole —CH(OH)—COOH benzyl 2 tetrazole —CH₂—COOH benzyl3 —SO₂NHC(O)CH₃ —CH(OH)—COOH benzyl 4 —SO₂NHC(O)OCH₃ —CH(OH)—COOH benzyl

Example 24

Following the procedures described in the examples above, andsubstituting the appropriate starting materials and reagents, compound24-1 to 24-4 having the following formula can also be prepared:

# R¹ R⁴ R⁵ 1 tetrazole —CH₂—COOH 2-Cl-benzyl 2 tetrazole —CH₂—COOH2-CF₃-benzyl 3 tetrazole —CH₂—COOH 2-F-benzyl 4 tetrazole —CH(OH)—COOHbenzyl 5 —SO₂NHC(O)CH₃ —CH(OH)—COOH benzyl

Assay 1 A. AT₁ and AT₂ Radioligand Binding Assays

These in vitro assays were used to assess the ability of test compoundsto bind to the AT₁ and the AT₂ receptors.

Membrane Preparation from Cells Expressing Human AT₁ or AT₂ Receptors

Chinese hamster ovary (CHO-K1) derived cell lines stably expressing thecloned human AT₁ or AT₂ receptors, respectively, were grown in HAM's-F12 medium supplemented with 10% fetal bovine serum, 10 μg/mlpenicillin/streptomycin, and 500 μg/ml geneticin in a 5% CO₂ humidifiedincubator at 37° C. AT₂ receptor expressing cells were grown in theadditional presence of 100 nM PD123,319 (AT₂ antagonist). When culturesreached 80-95% confluence, the cells were washed thoroughly in PBS andlifted with 5 mM EDTA. Cells were pelleted by centrifugation and snapfrozen in MeOH-dry ice and stored at −80° C. until further use.

For membrane preparation, cell pellets were resuspended in lysis buffer(25 mM Tris/HCl pH 7.5 at 4° C., 1 mM EDTA, and one tablet of CompleteProtease Inhibitor Cocktail Tablets with 2 mM EDTA per 50 mL buffer(Roche cat. #1697498, Roche Molecular Biochemicals, Indianapolis, Ind.))and homogenized using a tight-fitting Dounce glass homogenizer (10strokes) on ice. The homogenate was centrifuged at 1000×g, thesupernatant was collected and centrifuged at 20,000×g. The final pelletwas resuspended in membrane buffer (75 mM Tris/HCl pH 7.5, 12.5 mMMgCl₂, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose at 4° C.) and homogenizedby extrusion through a 20 G gauge needle. Protein concentration of themembrane suspension was determined by the method described in Bradford(1976) Anal Biochem. 72:248-54. Membranes were snap frozen in MeOH-dryice and stored at −80° C. until further use.

Ligand Binding Assay to Determine Compound Affinities for the Human AT₁and AT₂ Angiotensin Receptors

Binding assays were performed in 96-well Acrowell filter plates (PallInc., cat. #5020) in a total assay volume of 100 μL with 0.2 μg membraneprotein for membranes containing the human AT₁ receptor, or 2 μgmembrane protein for membranes containing the human AT₂ receptor inassay buffer (50 mM Tris/HCl pH 7.5 at 20° C., 5 mM MgCl₂, 25 μM EDTA,0.025% BSA). Saturation binding studies for determination of K_(d)values of the ligand were done using N-terminally Europium-labeledangiotensin-II ([Eu]AngII,H—(Eu—N¹)-Ahx-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH; PerkinElmer, Boston,Mass.) at 8 different concentrations ranging from 0.1 nM to 30 nM.Displacement assays for determination of pK_(i) values of test compoundswere done with [Eu]AngII at 2 nM and 11 different concentrations of drugranging from 1 pM to 10 μM. Drugs were dissolved to a concentration of 1mM in DMSO and from there serially diluted into assay buffer.Non-specific binding was determined in the presence of 10 μM unlabeledangiotensin-II. Assays were incubated for 120 minutes in the dark, atroom temperature or 37° C., and binding reactions were terminated byrapid filtration through the Acrowell filter plates followed by threewashes with 200 μL ice cold wash buffer (50 mM Tris/HCl pH 7.5 at 4° C.,5 mM MgCl₂) using a Waters filtration manifold. Plates were tapped dryand incubated with 50 μl DELFIA Enhancement Solution (PerkinElmer cat.#4001-0010) at room temperature for 5 minutes on a shaker. Filter-bound[Eu]AngII was quantitated immediately on a Fusion plate reader(PerkinElmer) using Time Resolved Fluorescence (TRF). Binding data wereanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using the3-parameter model for one-site competition. The BOTTOM (curve minimum)was fixed to the value for nonspecific binding, as determined in thepresence of 10 μM angiotensin II. K_(i) values for drugs were calculatedfrom observed IC₅₀ values and the K_(d) value of [Eu]AngII according tothe Cheng-Prusoff equation described in Cheng et al. (1973) BiochemPharmacol. 22(23):3099-108. Selectivities of test compounds for the AT₁receptor over the AT₂ receptor were calculated as the ratio ofAT₂K_(i)/AT₁K_(i). Binding affinities of test compounds were expressedas negative decadic logarithms of the K_(i) values (pK_(i)).

In this assay, a higher pK_(i) value indicates that the test compoundhas a higher binding affinity for the receptor tested.

B. In vitro Assays for the Quantitation of Inhibitor Potencies (IC₅₀) atHuman and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat NEP and humanACE were determined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold PBS and brought up in ice-cold lysisbuffer (1% Triton X-114, 150 mM NaCl, 50 mM Tris pH 7.5; Bordier (1981)J. Biol. Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for everygram of kidney. Samples were homogenized using a polytron hand heldtissue grinder on ice. Homogenates were centrifuged at 1000×g in aswinging bucket rotor for 5 minutes at 3° C. The pellet was resuspendedin 20 mL of ice cold lysis buffer and incubated on ice for 30 minutes.Samples (15-20 mL) were then layered onto 25 mL of ice-cold cushionbuffer (6% w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, TritonX-114), heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in aswinging bucket rotor at room temperature for 3 minutes. The two upperlayers were aspirated off, leaving a viscous oily precipitate containingthe enriched membrane fraction. Glycerol was added to a concentration of50% and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with BSA as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrate Mca-BK2(Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH; Johnson et al. (2000)Anal. Biochem. 286: 112-118) was used for the human NEP and ACE assays,and Mca-RRL (Mca-DArg-Arg-Leu-(Dnp)-OH; Medeiros et al. (1997) Braz. J.Med. Biol. Res. 30:1157-1162) was used for the rat NEP assay (both fromAnaspec, San Jose, Calif.).

The assays were performed in 384-well white opaque plates at roomtemperature using the respective fluorogenic peptides at a concentrationof 10 μM in assay buffer (50 mM Tris/HCl at 25° C., 100 mM NaCl, 0.01%Tween-20, 1 μM Zn, 0.025% BSA). Human NEP and human ACE were used atconcentrations that resulted in quantitative proteolysis of 5 μM ofMca-BK2 within 20 minutes at room temperature. The rat NEP enzymepreparation was used at a concentration that yielded quantitativeproteolysis of 3 μM of Mca-RRL within 20 minutes at room temperature.

Test compounds were diluted to 12 concentrations from 10 μM to 20 pM inAssay Buffer. Assays were started by adding 25 μL of enzyme to 12.5 μLof test compound at each of the 12 concentrations. Test compounds wereallowed to equilibrate with the enzyme for 10 minutes before 12.5 μL ofthe fluorogenic substrates were added to initiate the reaction.Reactions were terminated by the addition of 10 μL of 3.6% glacialacetic acid after 20 minutes of incubation.

For sulfhydryl-containing test compounds, the test compounds may bediluted in Assay Buffer containing a 400 μM concentration oftris(2-carboxyethyl)phosphine hydrochloride (Thermo Scientific,Rockford, Ill.) (TCEP). The test compounds are then incubated for 40minutes at room temperature (to allow for dimer cleavage) before addingthe enzyme. Test compounds are then allowed to equilibrate with theenzyme for 20 minutes before adding the fluorogenic substrates.Reactions are terminated as above.

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively. Raw data (relativefluorescence units) were normalized to % activity from the average highreadings (no inhibition, 100% enzyme activity) and average low readings(full inhibition, highest inhibitor concentration, 0% enzyme activity)using three standard NEP and ACE inhibitors, respectively. Nonlinearregression of the normalized data was performed using a one sitecompetition model (GraphPad Software, Inc., San Diego, Calif.). Datawere reported as pIC₅₀ values.

Compounds of the invention were tested in these or similar assays andwere found to have pK_(i) values at the AT₁ receptor and pIC₅₀ valuesfor the NEP enzyme as set forth below.

AT₁ NEP Ex. pK_(i) pIC₅₀ 1 ≧8.0 ≧6.0 2 ≧8.0 ≧5.0 3-1  ≧8.0 ≧7.0 3-2 ≧8.0 ≧7.0 3-3  ≧7.0 ≧7.0 3-4  ≧8.0 ≧7.0 3-5  ≧7.0 ≧7.0 3-6  ≧8.0 ≧7.03-7  ≧8.0 ≧7.0 3-8  ≧7.0 ≧6.0 3-9  ≧7.0 ≧6.0 3-10 ≧8.0 ≧6.0 3-11 ≧8.0≧7.0 3-12 ≧8.0  ≧5.0* 3-13 ≧8.0 ≧6.0 3-14 ≧8.0 ≧7.0 3-15 ≧8.0 ≧7.0 3-16≧8.0 ≧7.0 3-17 ≧8.0 ≧7.0 3-18 ≧8.0 ≧6.0 3-19 ≧7.0 ≧6.0 3-20 ≧7.0 ≧6.03-21 ≧7.0 ≧6.0 3-22 ≧7.0 ≧7.0 3-23 ≧8.0 ≧7.0 3-24 ≧8.0 ≧6.0 3-25 ≧8.0≧7.0 3-26 ≧7.0 ≧7.0 3-27 ≧8.0  ≧5.0* 3-28 ≧8.0  ≧5.0* 3-29 ≧8.0  ≧5.0*3-30 ≧8.0 ≧5.0 3-31 ≧7.0 ≧5.0 3-32 ≧7.0 ≧5.0 3-33 ≧8.0 ≧5.0 3-34 ≧8.0≧5.0 3-35 ≧8.0 ≧5.0 3-36 ≧7.0 ≧6.0 3-37 ≧7.0 ≧5.0 3-38 ≧7.0 ≧7.0 4 ≧8.0≧7.0 5 ≧8.0 ≧7.0 6-1  ≧8.0 ≧7.0 6-2  ≧8.0 ≧7.0 6-3  ≧7.0 ≧6.0 6-4  ≧8.0≧7.0 6-5  ≧8.0 ≧7.0 6-6  ≧8.0 ≧7.0 6-7  ≧8.0 ≧7.0 6-8  ≧8.0 ≧6.0 6-9 ≧8.0 ≧7.0 6-10 ≧7.0 ≧7.0 6-11 ≧8.0 ≧6.0 6-12 ≧8.0 ≧6.0 6-13 ≧8.0 ≧7.06-14 ≧8.0 ≧7.0 6-15 ≧8.0 ≧7.0 6-16 ≧8.0 ≧7.0 6-17 ≧8.0 ≧7.0 6-18 ≧8.0≧7.0 6-19 ≧8.0 ≧7.0 6-20 ≧8.0 ≧7.0 6-21 ≧8.0 ≧7.0 6-22 ≧8.0 ≧7.0 6-23≧8.0 ≧7.0 6-24 ≧8.0 ≧5.0 6-25 ≧8.0 ≧6.0 6-26 ≧8.0 ≧6.0 6-27 ≧8.0 ≧6.06-28 ≧8.0 ≧5.0 7-1  ≧8.0 ≧6.0 7-2  ≧8.0 ≧7.0 7-3  ≧8.0 ≧6.0 7-4  ≧8.0≧6.0 7-5  ≧8.0 ≧6.0 7-6  ≧8.0 ≧6.0 7-7  ≧8.0 ≧6.0 7-8  ≧8.0 ≧6.0 7-9 ≧8.0 ≧6.0 7-10 ≧8.0 ≧7.0 7-11 ≧8.0 ≧7.0 7-12 ≧8.0 ≧6.0 7-13 ≧8.0 ≧7.07-14 ≧8.0 ≧7.0 7-15 ≧8.0 ≧7.0 7-16 ≧8.0 ≧7.0 7-17 ≧8.0 ≧7.0 7-18 ≧8.0≧6.0 7-19 ≧7.0 ≧6.0 7-20 ≧8.0 ≧7.0 7-21 ≧7.0 ≧7.0 7-22 ≧7.0 ≧7.0 8 ≧7.0≧7.0 9-1  ≧7.0 ≧7.0 9-2  ≧7.0 ≧7.0 9-3  ≧7.0 ≧7.0 9-4  ≧7.0 ≧7.0 9-5 ≧7.0 ≧7.0 9-6  ≧7.0 ≧7.0 9-7  ≧7.0 ≧7.0 10  ≧8.0 ≧7.0 11  ≧8.0 ≧7.0 12 ≧8.0 ≧7.0 13  ≧7.0 ≧7.0 14-1  ≧8.0 ≧7.0 14-2  ≧8.0 ≧7.0 14-3  ≧7.0 ≧7.014-4  ≧8.0 ≧7.0 14-5  ≧8.0 ≧7.0 14-6  ≧8.0 ≧7.0 14-7  ≧8.0 ≧7.0 14-8 ≧8.0 ≧6.0 14-9  ≧8.0 ≧7.0 14-10  ≧8.0 ≧7.0 14-11  ≧8.0 ≧6.0 14-12  ≧8.0≧7.0 14-13  ≧8.0 ≧7.0 14-14  ≧8.0 ≧6.0 14-15  ≧8.0 ≧6.0 15-1  ≧8.0 ≧6.015-2  ≧7.0 ≧6.0 15-3  ≧7.0 ≧7.0 15-4  ≧7.0 ≧6.0 16-1  ≧8.0 ≧7.0 16-2 ≧8.0 ≧6.0 16-3  ≧8.0 ≧7.0 16-4  ≧8.0 ≧6.0 16-5  ≧8.0 ≧6.0 16-6  ≧7.0≧7.0 16-7  ≧8.0 ≧7.0 15-1  ≧8.0 ≧6.0 17-2  ≧8.0 ≧6.0 17-3  ≧8.0 ≧7.017-4  ≧8.0 ≧6.0 17-5  ≧7.0 ≧7.0 17-6  ≧8.0 ≧6.0 18  ≧7.0 ≧6.0 *Thesecompounds are prodrugs and therefore may not exhibit NEP activity in anin vitro assay.

The compounds of Examples 19, 20, 21, and 22 are prodrugs of thecompounds of Examples 3-16, 10, 12, and 14-6, respectively, and were nottested in this assay. Assuming full conversion of the prodrug to theactive compound, the prodrugs arc expected to have similar in vitro andin vivo AT₁ activity and equivalent or better in vivo NEP activity astheir active counterpart.

Assay 2 Pharmacodynamic (PD) Assay for ACE, AT₁, and NEP Activity inAnesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg(i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery(PE 50 tubing) and bladder (URI-1 urinary silicone catheter) arccannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge)to faciliate spontaneous respiration. The animals are then allowed a 60minute stablization period and kept continuously infused with 5 mL/kg/hof saline (0.9%) throughout, to keep them hydrated and ensure urineproduction. Body temperature is maintained throughout the experiment byuse of a heating pad. At the end of the 60 minute stabilization period,the animals are dosed intravenously (i.v.) with two doses of angiotensin(AngI, 1.0 μg/kg, for ACE inhibitor activity; AngII, 0.1 μg/kg, for AT₁receptor antagonist activity) at 15 minutes apart. At 15 minutespost-second dose of angiotensin (AngI or AngII), the animals are treatedwith vehicle or test compound. Five minutes later, the animals arcadditionally treated with a bolus i.v. injection of atrial natriureticpeptide (ANP; 30 μg/kg). Urine collection (into pre-weighted eppendorftubes) is started immediately after the ANP treatment and continued for60 minutes. At 30 and 60 minutes into urine collection, the animals arere-challenged with angiotensin (AngI or AngII). Blood pressuremeasurements are done using the Notocord system (Kalamazoo, Mich.).Urine samples are frozen at −20° C. until used for the cGMP assay. UrinecGMP concentrations are determined by Enzyme Immuno Assay using acommercial kit (Assay Designs, Ann Arbor, Mich., Cat. No. 901-013).Urine volume is determined gravimetrically. Urinary cGMP output iscalculated as the product of urine output and urine cGMP concentration.ACE inhibition or AT_(i) antagonism is assessed by quantifying the %inhibition of pressor response to AngI or AngII, respectively. NEPinhibition is assessed by quantifying the potentiation of ANP-inducedelevation in urinary cGMP output.

Assay 3 In Vivo Evaluation of Antihypertensive Effects in the ConsciousSHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed aminimum of 48 hours acclimation upon arrival at the testing site. Sevendays prior to testing, the animals are either placed on a restrictedlow-salt diet with food containing 0.1% of sodium for sodium depletedSHRs (SD-SHR) or are placed on a normal diet for sodium repleted SHRs(SR-SHR). Two days prior to testing, the animals are surgicallyimplemented with catheters into a carotid artery and the jugular vein(PE50 polyethylene tubing) connected via a PE10 polyethylene tubing to aselected silicone tubing (size 0.020 ID×0.037 OD×0.008 wall) for bloodpressure measurement and test compound delivery, respectively. Theanimals are allowed to recover with appropriate post operative care. Onthe day of the experiment, the animals are placed in their cages and thecatheters are connected via a swivel to a calibrated pressuretransducer. After 1 hour of acclimation, a baseline measurement is takenover a period of at least five minutes. The animals are then dosed i.v.with vehicle or test compound in ascending cumulative doses every 60minutes followed by a 0.3 mL saline to clear the catheter after eachdose. Data is recorded continuously for the duration of the study usingNotocord software (Kalamazoo, Mich.) and stored as electronic digitalsignals. In some studies, the effects of a single intravenous or oral(gavage) dose are monitored for at least 6 hours after dosing.Parameters measured are blood pressure (systolic, diastolic and meanarterial pressure) and heart rate.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) areallowed a minimum of 48 hours acclimation upon arrival at the testingsite before they are placed on a high salt diet. One week after thestart of the high salt diet, a DOCA-salt pellet (100 mg, 21 days releasetime, Innovative Research of America, Sarasota, Fla.) is implantedsubcutaneously and unilateral nephrectomy is performed. On 16 or 17 dayspost DOCA-salt pellet implantation, animals are implanted surgicallywith catheters into a carotid artery and the jugular vein with a PE50polyethylene tubing, which in turn was connected via a PE10 polyethylenetubing to a selected silicone tubing (size 0.020 ID×0.037 OD×0.008 wall)for blood pressure measurement and test compound delivery, respectively.The animals are allowed to recover with appropriate post operative care.

On the day of the experiment, each animal is kept in its cage andconnected via a swivel to a calibrated pressure transducer. After 1 hourof acclimation, a baseline measurement is taken over a period of atleast five minutes. The animals are then dosed i.v. with a vehicle ortest compound in escalating cumulative doses every 60 minutes

1. A compound of formula I:

where: Z is

Ar is selected from:

R¹ is selected from —SO₂NHC(O)R^(1a), tetrazolyl, —C(O)OR^(1b),

where R^(1a) is —C₁₋₆alkyl, —C₀₋₆alkylene-OR, —C₃₋₇cycloalkyl,—C₀₋₅alkylene-NR^(1b)R^(1b), pyridyl, isoxazolyl, methylisoxazolyl,pyrrolidinyl, morpholinyl, or phenyl optionally substituted with halo;where each R^(1b) is independently selected from H and —C₁₋₆alkyl; a is0, 1, or 2; R² is F; R³ is selected from —C₂₋₅alkyl and —O—C₁₋₅alkyl; R⁴is selected from —CH₂—SR^(4a), —CH₂—N(OH)C(O)H,—CH(R^(4b))C(O)NH(OR^(4d)), and —CH(R^(4b))C(O)OR^(4c); where R^(4a) isH or —C(O)—C₁₋₆alkyl; R^(4b) is H or —OH; R^(4c) is H, —C₁₋₆alkyl,—C₀₋₆alkylenemorpholine, —CH₂OC(O)O—C₁₋₆alkyl, —CH(CH₃)OC(O)O—C₁₋₆alkyl,—CH(CH₃)OC(O)O—C₃₋₇cycloalkyl, or:

R^(4d) is H or —C(O)—R^(4e); and R^(4e) is —C₁₋₆alkyl, —C₁₋₆alkyl-NH₂ oraryl; and R⁵ is selected from —C₁₋₆alkyl, —CH₂-furanyl, —CH₂-thiophenyl,benzyl, and benzyl substituted with one or more halo, —CH₃, or —CF₃groups; where each ring in Ar is optionally substituted with 1 to 3substituents independently selected from —OH, —C₁₋₆alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, —CN, halo, —O—C₁₋₆alkyl, —S—C₁₋₆alkyl, —S(O)—C₁₋₆alkyl,—S(O)₂—C₁₋₄alkyl, -phenyl, —NO₂, —NH₂, —NH—C₁₋₆alkyl and —N(C₁₋₆alkyl)₂,wherein each alkyl, alkenyl and alkynyl is optionally substituted with 1to 5 fluoro atoms; or a pharmaceutically acceptable salt thereof. 2.(canceled)
 3. The compound of claim 1, where Ar is:


4. The compound of claim 1, where R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃,—SO₂NHC(O)OCH₃, —SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl, —COOH, —C(O)OCH₃,


5. The compound of claim 1, where R³ is propyl, ethyl, butyl, or ethoxy.6. The compound of claim 1, where R⁴ is —CH₂SH, —CH₂N(OH)C(O)H,—CH₂C(O)NH(OH), —CH(OH)C(O)NH(OH), —CH(OH)COOH, or —CH₂COOH.
 7. Thecompound of claim 1, where R⁴ is —CH₂—S—C(O)CH₃, —CH₂C(O)NH—OC(O)CH₃,—CH₂C(O)NH—OC(O)-phenyl, —CH₂C(O)NH—OC(O)—CH(NH₂)[CH(CH₃)₂],—CH(OH)C(O)OCH₃, —CH₂C(O)OCH₃, —CH₂C(O)OCH₂CH₃, —CH₂C(O)OCH(CH₃)₂,—CH₂C(O)O(CH₂)₂CH₃, —CH₂C(O)O(CH₂)₃CH₃, —CH₂C(O)O(CH₂)₄CH₃,—CH₂C(O)OCH(CH₃)OC(O)OCH₂CH₃, —CH₂C(O)OCH(CH₃)OC(O)OCH(CH₃)₂,—CH₂C(O)OCH(CH₃)OC(O)O-cyclohexyl,


8. The compound of claim 1, where R⁵ is i-butyl, —CH₂-furan-2-yl,—CH₂-thiophen-3-yl, benzyl, 2-bromobenzyl, 2-chlorobenzyl,2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2-methylbenzyl, or2-trifluoromethylbenzyl.
 9. The compound of claim 1, wherein: Ar is:

R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃, —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl, —COOH, —C(O)OCH₃,

R³ is propyl, ethyl, butyl, or ethoxy; R⁴ is —CH₂SH, —CH₂—S—C(O)CH₃,—CH₂N(OH)C(O)H, —CH₂C(O)NH(OH), —CH₂C(O)NH—OC(O)CH₃,—CH₂C(O)NH—OC(O)-phenyl, —CH₂C(O)NH—OC(O)—CH(NH₂)[CH(CH₃)₂],—CH(OH)C(O)NH(OH), —CH(OH)COOH, —CH(OH)C(O)OCH₃, —CH₂COOH, —CH₂C(O)OCH₃,—CH₂C(O)OCH₂CH₃, —CH₂C(O)OCH(CH₃)₂, —CH₂C(O)O(CH₂)₂CH₃,—CH₂C(O)O(CH₂)₃CH₃, —CH₂C(O)O(CH₂)₄CH₃, —CH₂C(O)OCH(CH₃)OC(O)OCH₂CH₃,—CH₂C(O)OCH(CH₃)OC(O)OCH(CH₃)₂, —CH₂C(O)OCH(CH₃)OC(O)O-cyclohexyl,

and R⁵ is i-butyl, —CH₂-furan-2-yl, —CH₂-thiophen-3-yl, benzyl,2-bromobenzyl, 2-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl,4-fluorobenzyl, 2-methylbenzyl, or 2-trifluoromethylbenzyl.
 10. Thecompound of claim 1, having formula IV:


11. The compound of claim 10 where Ar is:

R¹ is —SO₂NHC(O)CH₃, —SO₂NHC(O)CH₂CH₃, —SO₂NHC(O)OCH₃,—SO₂NHC(O)OCH₂CH₃, —SO₂NHC(O)CH₂OCH₃, —SO₂NHC(O)CH₂OH,—SO₂NHC(O)CH(CH₃)OH, —SO₂NHC(O)C(CH₃)₂OH, —SO₂NHC(O)CH₂OCH₃,—SO₂NHC(O)(CH₂)₂OCH₃, —SO₂NHC(O)-cyclopropyl, —SO₂NHC(O)NH(CH₃),—SO₂NHC(O)N(CH₃)₂, —SO₂NHC(O)NH(CH₂CH₃), —SO₂NHC(O)C(CH₃)₂NH₂,—SO₂NHC(O)-2-pyridyl, —SO₂NHC(O)-4-pyridyl, —SO₂NHC(O)-5-isoxazolyl,—SO₂NHC(O)-3-isoxazolyl-5-methyl, —SO₂NHC(O)-1-pyrrolidyl,—SO₂NHC(O)-4-morpholinyl, —SO₂NHC(O)phenyl, —SO₂NHC(O)-2-fluorophenyl,1H-tetrazol-5-yl, —COOH, —C(O)OCH₃,

R³ is propyl, ethyl, butyl, or ethoxy; R^(4b) is H or —OH; and R^(4c) isH, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —(CH₂)₂CH₃, —(CH₂)₃CH₃, —(CH₂)₄CH₃,—CH(CH₃)OC(O)OCH₂CH₃, —CH(CH₃)OC(O)OCH(CH₃)₂, —CH(CH₃)OC(O)O-cyclohexyl,

and R⁵ is i-butyl, —CH₂-furan-2-yl, —CH₂-thiophen-3-yl, benzyl,2-bromobenzyl, 2-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl,4-fluorobenzyl, 2-methylbenzyl, or 2-trifluoromethylbenzyl. 12-16.(canceled)